JPH11164A - New phytase and its production - Google Patents
New phytase and its productionInfo
- Publication number
- JPH11164A JPH11164A JP9171223A JP17122397A JPH11164A JP H11164 A JPH11164 A JP H11164A JP 9171223 A JP9171223 A JP 9171223A JP 17122397 A JP17122397 A JP 17122397A JP H11164 A JPH11164 A JP H11164A
- Authority
- JP
- Japan
- Prior art keywords
- phytase
- optimum
- stability
- temperature
- activity
- 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.)
- Granted
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、新規フィターゼ、
これを生産する微生物及び該フィターゼの製造法に関す
る。更に詳細にはペニシリウム（Penicillium）属由来
の新規フィターゼに関し、飼料や食品の品質改善及び環
境汚染防止に利用することができ、飼料工業、食品工業
分野において有用である。TECHNICAL FIELD The present invention relates to a novel phytase,
The present invention relates to a microorganism producing the same and a method for producing the phytase. More specifically, the present invention relates to a novel phytase derived from the genus Penicillium , which can be used for improving the quality of feed and food and preventing environmental pollution, and is useful in the feed and food industries.
【０００２】[0002]
【従来の技術】リンは全ての生物の生育に必須の元素で
あるが、穀類、豆類等の植物種子に含有されているリン
の大部分はフィチン態で存在する。単胃動物であるヒト
や、豚、鶏等の家畜は、フィチンを分解できないため、
植物種子由来の食品や飼料（穀類、豆類）を摂取して
も、フィチン態リンを全く利用できない。そのため、単
胃家畜を良好に生育させるために、不足分のリン酸分と
してリン酸カルシウム等の無機リン酸を飼料中に添加す
ることが一般的に行われている。また、食品や飼料中の
フィチン態リンは、栄養源として利用されないだけでな
く、そのまま排泄物中に排出されるため、環境汚染（リ
ン公害）を引き起こし、問題となっている。さらに、フ
ィチン酸は、タンパク質と結合し、消化酵素の働きを阻
害させたり、カルシウム、亜鉛、マグネシウム、鉄等の
ミネラルをキレートし、栄養的に重要なミネラルの吸収
を妨げることから、栄養学的な面からフィチン酸の除去
方法が望まれている。2. Description of the Related Art Phosphorus is an essential element for the growth of all living organisms, but most of the phosphorus contained in plant seeds such as cereals and legumes exists in a phytin form. Domestic animals such as humans, pigs and chickens, which are monogastric animals, cannot decompose phytin,
Phytic phosphorus cannot be used at all even when foods and feeds (cereals and beans) derived from plant seeds are consumed. Therefore, in order to favorably grow monogastric livestock, it is common practice to add inorganic phosphoric acid such as calcium phosphate to the feed as an insufficient phosphate content. In addition, phytin phosphorus in foods and feeds is not only used as a nutrient source, but is also discharged directly into excretions, causing environmental pollution (phosphorus pollution), which is a problem. In addition, phytic acid binds to proteins and inhibits the action of digestive enzymes, chelates minerals such as calcium, zinc, magnesium, and iron, and interferes with the absorption of nutritionally important minerals. Therefore, a method for removing phytic acid has been desired.
【０００３】[0003]
【発明が解決しようとする課題】これらの問題を解決す
るため、微生物由来のフィターゼの利用、すなわち、食
品や飼料にフィターゼを添加することによりフィチン酸
を分解せしめ、ミネラルの吸収阻害を防止し、また排泄
されるフィチン態リンの量を低減させ、環境汚染を防止
することが報告されている（J.Nutrition,101,1289(197
1)等）。In order to solve these problems, the use of phytase derived from microorganisms, that is, the addition of phytase to foods and feeds, degrades phytic acid and prevents the inhibition of mineral absorption, In addition, it has been reported that the amount of excreted phytin phosphorus is reduced to prevent environmental pollution (J. Nutrition, 101 , 1289 (197
1) etc.).
【０００４】フィターゼ生産菌としてはアスペルギルス
（Aspergillus）属、ノイロスポラ（Neurospora）属等
のかび、サッカロミセス・セレビシエ（Saccharomyces
cerevisiae）等の酵母、バチラス（Bacillus）属、シュ
ウドモナス（Pseudomonas）属等の細菌が知られてい
る。しかしながら、これまで知られた微生物の多くはフ
ィターゼ生産能が非常に低く、酵素製造コストが高くな
るため応用はほとんど進んでいない。また、フィターゼ
生産能が比較的高いものとして、ノイロスポラ（Neuros
pora）属（特開平7-59562）やペニシリウム・カゼイコ
ラム（Penicillium caseicolumn）（特開平7-67635）が
報告されているが、飼料や食品にフィターゼを直接添加
し消化器系で作用させるためには、中性から酸性領域で
安定であり且つ作用することが必要であるのに対し、前
者は酸性域での安定性に、後者は弱酸性〜中性域での安
定性に問題がある。本発明は、実用的に使用可能なフィ
ターゼ生産能の高い微生物により、中性から酸性域で安
定且つ良好に作用する新規なフィターゼを製造する方法
を提供しようとするものである。Examples of phytase-producing bacteria include molds of the genus Aspergillus and Neurospora , and Saccharomyces.
cerevisiae ) and bacteria such as Bacillus and Pseudomonas are known. However, most of the microorganisms known so far have very low phytase-producing ability and the cost for producing the enzyme is high, so that their application has hardly progressed. In addition, as a relatively high phytase production ability, Neurospora ( Neurospora)
PorA) genus (JP 7-59562) and Penicillium casei column (Penicillium Caseicolumn) (JP-A-7-67635) have been reported, in order to act in the addition of phytase to the feed or food directly digestive system Are required to be stable and act in a neutral to acidic range, whereas the former has a problem in stability in an acidic range, and the latter has a problem in stability in a weakly acidic to neutral range. An object of the present invention is to provide a method for producing a novel phytase that acts stably and satisfactorily in a neutral to acidic region by using a microorganism having a high phytase-producing ability that can be used practically.
【０００５】[0005]
【課題を解決するための手段】本発明者らは、前記のよ
うな性質を有するフィターゼ生産能の高い微生物を広く
自然界より探索した結果、土壌より分離したペニシリウ
ム（Penicillium）属に属する微生物が顕著に高いフィ
ターゼ生産能を有し、本菌の生産するフィターゼがかか
る用件を満たすことを見い出した。さらに、鋭意検討の
結果、本菌を用いてフィターゼを著量製造する方法を確
立すると共に、ペニシリウム属の菌株において、中性か
ら酸性域で安定且つ良好に作用するフィターゼ生産菌株
は本菌株が初めてであることを知ることによって本発明
を完成するに到った。すなわち本発明はペニシリウム属
に属する新規な微生物を培養することにより、その培養
液中に中性から酸性域で安定且つ良好に作用する特定の
新規フィターゼを生成蓄積せしめ、培養物から該フィタ
ーゼを採取することを可能にするものである。Means for Solving the Problems The inventors of the present invention have searched extensively from the natural world for microorganisms having the above-mentioned properties and high phytase-producing ability and found that microorganisms belonging to the genus Penicillium isolated from soil are remarkable. It was found that the phytase produced by the present bacterium satisfies such a requirement. Furthermore, as a result of intensive studies, we established a method for producing phytase in a significant amount using this bacterium, and this strain was the first phytase-producing strain that works stably and favorably in the neutral to acidic range in strains of the genus Penicillium. Thus, the present invention has been completed. That is, the present invention cultivates a novel microorganism belonging to the genus Penicillium, thereby producing and accumulating a specific novel phytase that acts stably and satisfactorily in a neutral to acidic region in the culture solution, and collecting the phytase from the culture. It is possible to do.
【０００６】−生産菌− 本発明のフィターゼの生産に使用する微生物は、ペニシ
リウム属に属し、上記の理化学的性質を有するフィター
ゼを生産することができるものであれば、いかなるもの
でも良いが、具体的には本発明者らが土壌より分離した
ペニシリウムＡ−１３４６株が挙げられる。本菌株は、
以下に示すような菌学的性質を有する。 （ａ）形態 分生子柄は単生で、滑面であり、長さ10〜58μｍ、幅は
約２μｍである。フィアライドは矛先型で、長さ10〜18
μｍ、幅２〜３μｍであり、１分生子柄当たり１〜３個
形成する。分生子は卵形〜楕円形で長さ４〜５μｍ、幅
２〜３μｍであり、フィアライド先端部に連鎖状に形成
する。 （ｂ）生育 ツァペックイーストエキス寒天培地で培養した場合は、
37℃、７日間で直径27〜32ｍｍ、25℃、７日間で直径26
〜28ｍｍの菌叢に生育する。集落表面の組織は薄いビロ
ード状であり、集落表面の色調は白色〜黄色がかった白
色、集落裏面の色調は灰色がかった黄色である。生育ｐ
Ｈ範囲は３〜７で最適ｐＨは５〜６、生育温度範囲は15
〜40℃で最適温度は２８〜３０℃である。上記の菌学的
性質に基づき文献〔Compendium of Soil Fungi, volume
１(1993)等〕を参考に検討した結果、この菌株はペニシ
リウム（Penicillium）属に属することが判明した。ま
た本菌株は工業技術院生命工学工業技術研究所に寄託番
号ＦＥＲＭ Ｐ−１５７４４として寄託されている。-Producing Bacteria- The microorganism used for producing the phytase of the present invention may be any microorganism as long as it belongs to the genus Penicillium and can produce phytase having the above physicochemical properties. Specifically, Penicillium A-1346 strain isolated from the soil by the present inventors can be mentioned. This strain is
It has mycological properties as shown below. (A) Morphology The conidiophores are solitary and smooth, 10 to 58 μm in length and about 2 μm in width. Fearride is pierced, 10-18 length
μm, width 2-3 μm, and 1-3 are formed per conidium. Conidia are oval to elliptical, 4-5 μm in length and 2-3 μm in width, and form in a chain at the tip of the phialide. (B) Growth When cultured on Tzapek yeast extract agar medium,
37 ° C, Diameter 27-32mm in 7 days, 25 ° C, Diameter 26 in 7 days
Grows in a flora of ~ 28mm. The texture of the settlement surface is light velvet, the color of the settlement surface is white to yellowish white, and the color of the back surface of the settlement is grayish yellow. Growth p
The H range is 3-7, the optimal pH is 5-6, and the growth temperature range is 15
At ~ 40 ° C, the optimum temperature is 28-30 ° C. The literature (Compendium of Soil Fungi, volume
1 (1993)], it was found that this strain belongs to the genus Penicillium . This strain has been deposited with the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-15744.
【０００７】[0007]
【発明の実施の形態】本発明のフィターゼを製造するに
あたっては、上記菌株を培養し、その培養物より採取す
ればよい。培地の栄養源としては、本菌が円滑に生育す
る限り特に限定されるものではなく、炭素源としては、
資化しうる炭素化合物、例えばグルコース、フラクトー
ス、マルトース、サッカロース、油脂、穀類などを使用
することができ、窒素源としては資化しうる窒素化合
物、またはこれを含有するものであればよく、例えば硫
酸アンモニウム、硝酸ナトリウム、各種ペプトン、酵母
エキス、肉エキス、大豆粉、コーンスティープリカーな
どを用いることができる。また、マグネシウム、カルシ
ウム、ナトリウム、カリウム等の無機塩や、無機、有機
微量栄養源を培地中に添加することができる。DESCRIPTION OF THE PREFERRED EMBODIMENTS In producing the phytase of the present invention, the above strain may be cultured and collected from the culture. The nutrient source of the medium is not particularly limited as long as the bacterium grows smoothly, and as a carbon source,
Assimilable carbon compounds, for example, glucose, fructose, maltose, saccharose, fats and oils, grains and the like can be used.As the nitrogen source, any assimilable nitrogen compound or any containing it may be used, such as ammonium sulfate, Sodium nitrate, various peptones, yeast extract, meat extract, soybean powder, corn steep liquor and the like can be used. In addition, inorganic salts such as magnesium, calcium, sodium, and potassium, and inorganic and organic trace nutrients can be added to the medium.
【０００８】培養の形態は、液体、固体培養いずれでも
良い。液体培養における培養条件は培地組成により多少
異なるが、培地の初発ｐＨを３〜７の範囲、好ましくは
５〜６の範囲に調整し、培養温度15〜40℃、好ましくは
28〜30℃で好気的に３〜７日間程度培養を行い、フィタ
ーゼ生産量が最大に達したときに培養を終了すれば良
い。この様な培養により目的とするフィターゼは主とし
て菌体外に得られる。[0008] The form of culture may be either liquid or solid culture. The culture conditions in the liquid culture vary somewhat depending on the medium composition, but the initial pH of the medium is adjusted to a range of 3 to 7, preferably 5 to 6, and the culture temperature is 15 to 40 ° C, preferably
Culture may be performed aerobically at 28 to 30 ° C. for about 3 to 7 days, and the culture may be terminated when the phytase production reaches the maximum. By such a culture, the desired phytase is mainly obtained outside the cells.
【０００９】こうして得られた培養液から、目的物であ
るフィターゼを回収並びに精製するには、一般的に行わ
れる酵素の採取法ないし精製方法に準じて行えばよい。
すなわち、得られた培養液は、遠心分離や濾過等によっ
て菌体を除去した後、その上澄液を粗酵素液として回収
する。この粗酵素液はそのまま使用することもできる
が、必要に応じて硫安等の塩類による塩析法、あるいは
エタノール、アセトン等の有機溶剤による沈澱法等によ
って活性画分を回収し、限外濾過膜法等の常法により濃
縮後、イオン交換クロマトグラフィー、ゲル濾過等の精
製手段を適宜組み合わせてフィターゼを分別精製するこ
とができる。また、所望により適宜な乾燥法により、粉
末状に回収することもできる。The target phytase can be recovered and purified from the thus obtained culture broth by a general method of collecting or purifying the enzyme.
That is, the obtained culture solution is subjected to centrifugation, filtration and the like to remove the cells, and then the supernatant is recovered as a crude enzyme solution. This crude enzyme solution can be used as it is, but if necessary, the active fraction is recovered by salting out with a salt such as ammonium sulfate or the like, or precipitation with an organic solvent such as ethanol or acetone. After concentration by a conventional method such as a method, phytase can be fractionated and purified by appropriately combining purification means such as ion exchange chromatography and gel filtration. If desired, the powder can be recovered by a suitable drying method.
【００１０】−酵素活性測定法− 本発明において使用したフィターゼ活性の測定方法は以
下に示すとおりである。即ち、4mMフィチン酸ナトリウ
ムを含む 0.2M酢酸緩衝液（pH 5.5）0.5ml、蒸留水 0.4
ml及び酵素液 0.1mlからなる反応液を 37℃で 30分間反
応させ、10％ＴＣＡ 1.0mlを加えて反応を停止させる。
この反応液中の無機リン酸量をFiske-Subbarow法により
定量し、活性を求めた。活性の単位は、１分間に１マイ
クロモルの無機リン酸を遊離させる酵素量を１単位
（Ｕ）とした。-Method for measuring enzyme activity- The method for measuring phytase activity used in the present invention is as follows. That is, 0.5 ml of 0.2 M acetate buffer (pH 5.5) containing 4 mM sodium phytate, 0.4 ml of distilled water
A reaction solution consisting of 0.1 ml of the enzyme solution and 0.1 ml of the enzyme solution is reacted at 37 ° C. for 30 minutes, and the reaction is stopped by adding 1.0 ml of 10% TCA.
The amount of inorganic phosphoric acid in this reaction solution was quantified by the Fiske-Subbarow method to determine the activity. The unit of activity was 1 unit (U) of the amount of the enzyme that released 1 micromol of inorganic phosphoric acid per minute.
【００１１】−酵素学的性質− ペニシリウムＡ−１３４６株の生産するフィターゼの酵
素学的性質は、以下のとおりである。 （１）作用：フィチン酸を加水分解して、ミオイノシト
ール及び遊離のリン酸を生成する。 （２）基質特異性：種々の基質に対する活性を測定し、
フィチン酸ナトリウムに対する活性を１００として表１
に示した。本発明のフィターゼは種々の基質に作用し、
特にフィチン酸及びｐ−ニトロフェニルリン酸に対して
よく作用した。-Enzymatic properties- The enzymatic properties of the phytase produced by Penicillium A-1346 strain are as follows. (1) Action: Hydrolyzes phytic acid to produce myo-inositol and free phosphoric acid. (2) Substrate specificity: activities for various substrates were measured,
Table 1 with the activity for sodium phytate as 100
It was shown to. The phytase of the present invention acts on various substrates,
Particularly, it acted well on phytic acid and p-nitrophenyl phosphate.
【００１２】[0012]
【表１】 [Table 1]
【００１３】（３）作用ｐＨ及び至適ｐＨ：フィチン酸
ナトリウムを基質とした際のｐＨの影響を図１に示し
た。本発明のフィターゼの作用ｐＨ範囲は２〜６であ
り、至適ｐＨは４〜５であった。 （４）ｐＨ安定性：各ｐＨ値において、３７℃にて１時
間処理した際のｐＨ安定性を図２に示した。本発明のフ
ィターゼは、ｐＨ２．５〜６．５の範囲で安定であっ
た。 （５）作用温度及び至適温度：フィチン酸ナトリウムを
基質とした際の、0.1M酢酸緩衝液（pH 5.5）中における
作用温度を図３に示した。本発明のフィターゼの作用温
度は３０〜６０℃であり、至適温度は５５℃であった。 （６）温度安定性：0.1M酢酸緩衝液（pH 5.5）中で、３
０分間処理した際の温度安定性を図４に示した。本発明
のフィターゼは５５℃まで安定であった。 （７）分子量：ゲル濾過法により得られた分子量は、約
130〜150kDaであった。 （８）等電点：ショ糖密度勾配等電点電気泳動法により
得られた等電点は、４．４であった。 （９）金属イオンの影響：活性測定時に１mMの各種金属
イオンを共存させた際の活性発現率を表２に示した。本
発明のフィターゼの活性は、Ｃｕ2+、Ｚｎ2+、Ｃｄ2+、
Ｈｇ2+、Ｓｎ2+、Ｐｂ2+、Ａｌ3+により阻害された。(3) Working pH and optimum pH: The effect of pH when sodium phytate is used as a substrate is shown in FIG. The working pH range of the phytase of the present invention was 2 to 6, and the optimum pH was 4 to 5. (4) pH stability: pH stability after treatment at 37 ° C. for 1 hour at each pH value is shown in FIG. The phytase of the present invention was stable in the pH range of 2.5 to 6.5. (5) Action temperature and optimal temperature: FIG. 3 shows the action temperature in 0.1 M acetate buffer (pH 5.5) when sodium phytate was used as a substrate. The working temperature of the phytase of the present invention was 30-60 ° C, and the optimum temperature was 55 ° C. (6) Temperature stability: 0.1 M acetate buffer (pH 5.5)
FIG. 4 shows the temperature stability when treated for 0 minutes. The phytase of the present invention was stable up to 55 ° C. (7) Molecular weight: The molecular weight obtained by the gel filtration method is about
130-150 kDa. (8) Isoelectric point: The isoelectric point obtained by sucrose density gradient isoelectric focusing was 4.4. (9) Influence of metal ions: Table 2 shows the activity expression rate when 1 mM of various metal ions coexisted during the activity measurement. The activity of the phytase of the present invention is Cu 2+ , Zn 2+ , Cd 2+ ,
Inhibited by Hg 2+ , Sn 2+ , Pb 2+ , Al 3+ .
【００１４】[0014]
【表２】 [Table 2]
【００１５】（１０）各種阻害剤の影響：活性測定時に
５mMの各種キレート剤、各種ＳＨ酵素阻害剤及びアジ化
ナトリウムを共存させた際の活性発現率を表３に示し
た。本発明のフィターゼの活性は、これらの阻害剤にほ
とんど影響されなかった。(10) Influence of various inhibitors: Table 3 shows the activity expression rate when 5 mM of various chelating agents, various SH enzyme inhibitors and sodium azide coexist at the time of activity measurement. The activity of the phytase of the present invention was hardly affected by these inhibitors.
【００１６】[0016]
【表３】 [Table 3]
【００１７】[0017]
【実施例】次に実施例により本発明を詳細に説明する
が、これによって本発明は限定を受けるものではない。Next, the present invention will be described in detail with reference to examples, but the present invention is not limited by these examples.
【００１８】−実施例１− フィターゼ生産に従来よく用いられているアスペルギル
ス属の菌株を用いて、本菌株との生産性の比較を行っ
た。米ぬか10％、塩化アンモニウム0.5％、硫酸マグネ
シウム0.05％、塩化カリウム0.05％を含む液体培地20ml
を100mlの三角フラスコに入れ、121℃で20分間滅菌した
後、各菌株の胞子懸濁液を接種し、30℃、210rpmで５日
間、回転振盪培養した。この培養液を遠心分離し、その
上澄のフィターゼ活性を測定した。その結果を表４に示
した。本菌株はアスペルギルス属の菌株に比べて顕著に
高い生産性を示した。Example 1 A strain of the genus Aspergillus, which has been conventionally used for phytase production, was used to compare productivity with the present strain. Liquid culture medium containing rice bran 10%, ammonium chloride 0.5%, magnesium sulfate 0.05%, potassium chloride 0.05% 20ml
Was placed in a 100 ml Erlenmeyer flask, sterilized at 121 ° C. for 20 minutes, inoculated with a spore suspension of each strain, and cultured at 30 ° C. at 210 rpm for 5 days with rotary shaking. The culture was centrifuged, and the phytase activity of the supernatant was measured. Table 4 shows the results. This strain showed remarkably higher productivity than the strain of the genus Aspergillus.
【００１９】[0019]
【表４】 [Table 4]
【００２０】−実施例２− 実施例１の液体培地2.5Ｌを５Ｌ容ジャーファーメンタ
ーに入れ、121℃で20分間滅菌した後、予め30℃、３日
間振盪培養したペニシリウムＡ−１３４６株の前培養液
を接種し、30℃、500rpm、１vvmで５日間通気撹拌培養
した。この培養液を遠心分離により菌体を除去して、上
澄液を回収し、フィターゼ活性を測定したところ4.8Ｕ
／mlであった。この上澄液に硫酸アンモニウムを0.8飽
和となるまで添加して、沈澱する酵素を回収した後、再
溶解して限外濾過膜（旭化成製 分画分子量6000）によ
り脱塩、濃縮を行った。さらに、凍結乾燥を行い、比活
性257Ｕ／ｇの粗酵素粉末を約25ｇ得た。Example 2 2.5 L of the liquid medium of Example 1 was placed in a 5 L jar fermenter, sterilized at 121 ° C. for 20 minutes, and before shaking culture at 30 ° C. for 3 days before Penicillium A-1346 strain. The culture solution was inoculated, and aerated with stirring at 30 ° C., 500 rpm, and 1 vvm for 5 days. The culture was centrifuged to remove the cells, the supernatant was recovered, and the phytase activity was measured.
/ Ml. Ammonium sulfate was added to the supernatant until 0.8 saturation was reached, the precipitated enzyme was recovered, redissolved, desalted and concentrated using an ultrafiltration membrane (Asahi Kasei's molecular weight cut off 6000). Further, freeze-drying was performed to obtain about 25 g of a crude enzyme powder having a specific activity of 257 U / g.
【００２１】−実施例３− 各飼料成分（脱脂米糠、小麦ふすま、とうもろこし）１
ｇを水15mlに懸濁した後、本酵素を５Ｕ添加し、39℃で
60分間処理した（食道の条件）。次に、0.5％ペプシン
溶液（pH３）を５ml添加後、塩酸を用いてｐＨ３に調整
し、39℃で90分間処理した（胃の条件）。処理後の遊離
リン酸量を定量し、酵素無添加の条件での遊離リン酸量
を差し引くことにより、酵素反応による遊離リン酸量を
算出した。その結果を表５に示した。本酵素により各飼
料成分に含まれているフィチン酸が有効に分解されるこ
とが認められた。Example 3 Each feed component (defatted rice bran, wheat bran, corn) 1
g of the enzyme was suspended in 15 ml of water, and 5 U of the enzyme was added.
Treated for 60 minutes (esophageal conditions). Next, 5 ml of a 0.5% pepsin solution (pH 3) was added, the pH was adjusted to 3 with hydrochloric acid, and the mixture was treated at 39 ° C. for 90 minutes (stomach conditions). The amount of free phosphoric acid after the treatment was quantified, and the amount of free phosphoric acid due to the enzyme reaction was calculated by subtracting the amount of free phosphoric acid under the condition where no enzyme was added. Table 5 shows the results. It was confirmed that phytic acid contained in each feed component was effectively degraded by this enzyme.
【００２２】[0022]
【表５】 [Table 5]
【００２３】[0023]
【発明の効果】ペニシリウム属のカビを用いることによ
り、大量のフィターゼを容易且つ安価に取得できる。ま
た、本発明のフィターゼは、中性から酸性域で安定且つ
良好に作用するため、飼料や食品にフィターゼを直接添
加することにより、消化器官内でフィチンを分解し、遊
離したリン酸の有効利用、ミネラル吸収阻害防止、並び
に排泄物中のリン低減化による環境汚染防止等の効果を
有する。According to the present invention, a large amount of phytase can be easily and inexpensively obtained by using a fungus of the genus Penicillium. In addition, the phytase of the present invention acts stably and satisfactorily in a neutral to acidic region. Therefore, by directly adding phytase to feed or food, phytin is decomposed in the digestive tract, and the effective use of released phosphoric acid It has the effect of preventing the inhibition of mineral absorption and the prevention of environmental pollution by reducing phosphorus in excrement.
【図面の簡単な説明】[Brief description of the drawings]
【図１】 本発明の酵素の作用ｐＨ及び至適ｐＨの関係
を示す。縦軸の値は、pH4.5における活性を100％とした
相対活性である。FIG. 1 shows the relationship between the working pH and the optimum pH of the enzyme of the present invention. The values on the vertical axis are relative activities with the activity at pH 4.5 being 100%.
【図２】 本発明の酵素のｐＨ安定性を示す。縦軸の値
は、37℃にて１時間処理した後の残存活性である。FIG. 2 shows the pH stability of the enzyme of the present invention. The value on the vertical axis is the residual activity after treatment at 37 ° C. for 1 hour.
【図３】 本発明の酵素の作用温度及び至適温度の関係
を示す。縦軸の値は、55℃における活性を100％とした
相対活性である。FIG. 3 shows the relationship between the working temperature and the optimum temperature of the enzyme of the present invention. The values on the vertical axis are relative activities with the activity at 55 ° C. being 100%.
【図４】 本発明の酵素の温度安定性を示す。縦軸の値
は、pH 5.5にて30分間処理した後の残存活性である。FIG. 4 shows the temperature stability of the enzyme of the present invention. The value on the vertical axis is the residual activity after treatment at pH 5.5 for 30 minutes.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ｃ１２Ｒ 1:80） ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code FI C12R 1:80)
Claims (3)
ゼ。 (1)作用；フィチン酸を加水分解して、ミオイノシトー
ル及び遊離のリン酸を生成する。 (2)基質特異性；フィチン酸に対して強く作用し、各種
リン酸エステルにも作用する。 (3)作用ｐＨ及び至適ｐＨ；２〜６の範囲で活性を示
し、至適ｐＨは４〜５である。 (4)ｐＨ安定性；３７℃で１時間保持した場合、ｐＨ
２．５〜６．５の範囲で安定である。 (5)作用温度及び至適温度；３０〜６０℃の範囲で活性
を示し、至適温度は５５℃である。 (6)温度安定性；ｐＨ５．５で３０分間保持した場合、
５５℃まで安定である。 (7)分子量；ゲル濾過法による分子量は約130〜150kDaで
ある。 (8)等電点；ショ糖密度勾配等電点電気泳動法による等
電点は、４．４である。1. A novel phytase having the following physicochemical properties. (1) action; hydrolyze phytic acid to produce myo-inositol and free phosphoric acid. (2) Substrate specificity: Strongly acts on phytic acid and also acts on various phosphate esters. (3) action pH and optimum pH; activity is shown in the range of 2 to 6, and the optimum pH is 4 to 5. (4) pH stability; pH at 37 ° C. for 1 hour
It is stable in the range of 2.5 to 6.5. (5) Working temperature and optimum temperature: The activity is in the range of 30 to 60 ° C, and the optimum temperature is 55 ° C. (6) Temperature stability; when kept at pH 5.5 for 30 minutes,
Stable up to 55 ° C. (7) Molecular weight: The molecular weight by gel filtration is about 130 to 150 kDa. (8) Isoelectric point: The isoelectric point by sucrose density gradient isoelectric focusing is 4.4.
ニシリウム（Penicillium）属に属する微生物2. A microorganism belonging to the genus Penicillium which produces the phytase according to claim 1.
の天然ないし人工変異株を培養し、培養物中に請求項１
に記載のフィターゼを生成蓄積せしめ、該培養物中から
これを採取することを特徴とするフィターゼの製造法3. The novel microorganism strain according to claim 2, or a natural or artificial mutant thereof, is cultured and cultured in a culture.
A method for producing phytase, comprising producing and accumulating the phytase according to claim 1, and collecting the phytase from the culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17122397A JP3876046B2 (en) | 1997-06-13 | 1997-06-13 | Novel phytase and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17122397A JP3876046B2 (en) | 1997-06-13 | 1997-06-13 | Novel phytase and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH11164A true JPH11164A (en) | 1999-01-06 |
| JP3876046B2 JP3876046B2 (en) | 2007-01-31 |
Family
ID=15919336
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17122397A Expired - Fee Related JP3876046B2 (en) | 1997-06-13 | 1997-06-13 | Novel phytase and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3876046B2 (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4790995A (en) * | 1985-04-11 | 1988-12-13 | Scanflavour A/S | Process for sterilizng spices |
| WO2003066847A2 (en) | 2002-02-08 | 2003-08-14 | Novozymes A/S | Phytase variants |
| KR100414530B1 (en) * | 2000-09-30 | 2004-01-07 | 대한제당 주식회사 | Penicillium oxalicum |
| KR100470310B1 (en) * | 2002-02-07 | 2005-02-05 | 대한제당 주식회사 | An Anti-fungal Composition Comprising Phytase |
| JP2007300851A (en) * | 2006-05-11 | 2007-11-22 | Ichibiki Kk | Method for producing fermented feed containing soy sauce lees |
| WO2008017661A1 (en) | 2006-08-07 | 2008-02-14 | Novozymes A/S | Enzyme granules for animal feed |
| EP2143338A1 (en) | 2004-09-27 | 2010-01-13 | Novozymes A/S | Enzyme Granules |
| JP4965780B2 (en) * | 1999-08-13 | 2012-07-04 | ザ・ヴィクトリア・ユニバーシティ・オブ・マンチェスター | Phytase enzyme, nucleic acid encoding phytase enzyme, vector and host cell incorporating the above |
| WO2015197871A1 (en) | 2014-06-27 | 2015-12-30 | Dsm Ip Assets B.V. | A method for improving the nutritional value of animal feed |
| EP3072399A1 (en) | 2006-08-07 | 2016-09-28 | Novozymes A/S | Enzyme granules for animal feed |
| WO2021229093A1 (en) | 2020-05-15 | 2021-11-18 | Dsm Ip Assets B.V. | A method for improving the nutritional value of animal feed |
-
1997
- 1997-06-13 JP JP17122397A patent/JP3876046B2/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4790995A (en) * | 1985-04-11 | 1988-12-13 | Scanflavour A/S | Process for sterilizng spices |
| JP4965780B2 (en) * | 1999-08-13 | 2012-07-04 | ザ・ヴィクトリア・ユニバーシティ・オブ・マンチェスター | Phytase enzyme, nucleic acid encoding phytase enzyme, vector and host cell incorporating the above |
| KR100414530B1 (en) * | 2000-09-30 | 2004-01-07 | 대한제당 주식회사 | Penicillium oxalicum |
| KR100470310B1 (en) * | 2002-02-07 | 2005-02-05 | 대한제당 주식회사 | An Anti-fungal Composition Comprising Phytase |
| WO2003066847A2 (en) | 2002-02-08 | 2003-08-14 | Novozymes A/S | Phytase variants |
| EP2143338A1 (en) | 2004-09-27 | 2010-01-13 | Novozymes A/S | Enzyme Granules |
| EP2143339A1 (en) | 2004-09-27 | 2010-01-13 | Novozymes A/S | Enzyme Granules |
| DE202005021810U1 (en) | 2004-09-27 | 2010-04-22 | Novozymes A/S | Granules with a core and a coating |
| JP2007300851A (en) * | 2006-05-11 | 2007-11-22 | Ichibiki Kk | Method for producing fermented feed containing soy sauce lees |
| WO2008017661A1 (en) | 2006-08-07 | 2008-02-14 | Novozymes A/S | Enzyme granules for animal feed |
| EP3072399A1 (en) | 2006-08-07 | 2016-09-28 | Novozymes A/S | Enzyme granules for animal feed |
| WO2015197871A1 (en) | 2014-06-27 | 2015-12-30 | Dsm Ip Assets B.V. | A method for improving the nutritional value of animal feed |
| WO2021229093A1 (en) | 2020-05-15 | 2021-11-18 | Dsm Ip Assets B.V. | A method for improving the nutritional value of animal feed |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3876046B2 (en) | 2007-01-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR0169913B1 (en) | New strain bacillus sp.ds11 (kctc 0231bp)and new phytase produced from this | |
| CN101535467B (en) | A levorotatory lactonohydrolase producing strain and its use for producing chiral oxyacid | |
| JP3484208B2 (en) | Novel phytase and its production method | |
| US4062732A (en) | Process of producing acid stable protease | |
| JP3876046B2 (en) | Novel phytase and method for producing the same | |
| Kim et al. | Culture conditions for a new phytase-producing fungus | |
| JPH11113568A (en) | Enzyme complex | |
| Abu-Tahon et al. | Comparative study of a new alkaline L-methioninase production by Aspergillus ustus AUMC 10151 in submerged and solid-state fermentation | |
| IL34956A (en) | Production of lipase | |
| El-Fouly et al. | Biosynthesis and characterization of Aspergillus niger AUMC 4301 tannase | |
| JP3951008B2 (en) | Capsaicin decomposing / synthesizing enzyme and production method thereof | |
| JPH0253029B2 (en) | ||
| US6180390B1 (en) | Phytase-producing bacteria, phytase and production method of phytase | |
| HU181488B (en) | Process for the hydrolysis of racemic hydantoins into optically active n-carbamoyl-aminoacid derivatives and for preparing the hydrolyzing enzymatic complex | |
| JP3761236B2 (en) | Novel β-glucosidase, production method and use thereof | |
| US6033897A (en) | Phytase-producing bacteria | |
| Han | Use of microbial phytase in improving the feed quality of soya bean meal | |
| PARK et al. | Comparative enzymatic hydrolysis of phytate in various animal feedstuff with two different phytases | |
| KR0154398B1 (en) | Novel penicillum sp. am301 and phytase | |
| JP3917723B2 (en) | Lactone hydrolase and process for producing the same | |
| Joshi et al. | Production, purification and characterization of L-asparaginase from bacterial endophytes | |
| KR910007849B1 (en) | New microorganism stretomyces spy-183 | |
| JP3687406B2 (en) | Method for degrading chlorinated glycerides using microorganisms | |
| JPS5836953B2 (en) | Shinkinaripa Zenoseizouhou | |
| JPS6228678B2 (en) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20040528 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20060711 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20060831 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20060901 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20061024 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20061030 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20091102 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20101102 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111102 Year of fee payment: 5 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121102 Year of fee payment: 6 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121102 Year of fee payment: 6 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131102 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20141102 Year of fee payment: 8 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |