JPH0763364B2 - Method for producing raw starch degrading enzyme - Google Patents
Method for producing raw starch degrading enzymeInfo
- Publication number
- JPH0763364B2 JPH0763364B2 JP61202873A JP20287386A JPH0763364B2 JP H0763364 B2 JPH0763364 B2 JP H0763364B2 JP 61202873 A JP61202873 A JP 61202873A JP 20287386 A JP20287386 A JP 20287386A JP H0763364 B2 JPH0763364 B2 JP H0763364B2
- Authority
- JP
- Japan
- Prior art keywords
- degrading enzyme
- raw starch
- starch
- starch degrading
- raw
- 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.)
- Expired - Lifetime
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Description
【発明の詳細な説明】 発明の分野 本発明は、新規な生澱粉分解酵素の製造法に関する。Description: FIELD OF THE INVENTION The present invention relates to a method for producing a novel raw starch degrading enzyme.
発明の背景 澱粉粒を直接糖化することは、農産物の利用、醸造工程
の省力化、省エネルギーなどの点で有効である。この目
的には生澱粉分解酵素の使用が考えられるが、従来、好
適な酵素は見当たらない。Background of the Invention Direct saccharification of starch granules is effective in terms of utilization of agricultural products, labor saving in brewing process, energy saving and the like. The use of raw starch degrading enzyme is considered for this purpose, but hitherto no suitable enzyme has been found.
このような事情に鑑み、本発明者らは、土壌、花、果実
等より、数多くの微生物を分離し、その生澱粉分解活性
について鋭意研究を重ねた。その結果、単離された糸状
菌が生澱粉分解酵素を生産し、しかも、糊化澱粉分解活
性に対する生澱粉分解活性の比が非常に高い特徴を有す
ることを見出し、本発明を完成するにいたった。In view of such circumstances, the present inventors have separated a large number of microorganisms from soil, flowers, fruits, etc., and have conducted extensive studies on their raw starch degrading activity. As a result, it was found that the isolated filamentous fungus produces a raw starch degrading enzyme and has a very high ratio of the raw starch degrading activity to the gelatinized starch degrading activity, and the present invention was completed. It was
発明の開示 すなわち、本発明は新規な生澱粉分解酵素の製造法に関
するものである。DISCLOSURE OF THE INVENTION That is, the present invention relates to a novel method for producing a raw starch degrading enzyme.
本発明の新規生澱粉分解酵素生産糸状菌は次のような菌
学的性質を有するものである。The novel raw starch degrading enzyme-producing filamentous fungus of the present invention has the following mycological properties.
（１）生育状態 ツァペック液寒天（Czapek′s solution agar）倍地
を用い、28℃、２週間平板培養することにより、直径４
〜5cmのコロニーを生ずる。また、色はほとんどが白色
であり、コロニー中央部の一部で胞子形状が見られ淡緑
色となる。(1) Growing state Using Czapek's solution agar medium, plating at 28 ° C for 2 weeks, the diameter of 4
Gives ~ 5 cm colonies. In addition, most of the color is white, and a spore shape is seen in a part of the central part of the colony, and the color is pale green.
麦芽エキス寒天（Malt extract agar）倍地を用い、2
8℃、２週間平板培養することにより、直径4cm程度のコ
ロニーを生じ、ビロード状の生育を示す。色は緑色〜灰
緑色であり、非常によく胞子を形成する。分生子柄は気
菌糸から生じ、最大、長さは200μ、巾は３μである。
壁は滑面である。また、、フィアロ型分生子を作り、そ
の完全世代が認められない。Using Malt extract agar medium, 2
By plating at 8 ° C. for 2 weeks, colonies with a diameter of about 4 cm are produced and show velvety growth. The color is green to gray-green and it spores very well. Conidia peduncle arises from aerial hyphae, with a maximum length of 200μ and width of 3μ.
The wall is smooth. In addition, the Fiaro-type conidia are made and the perfect generation is not recognized.
ツァペック液寒天（Czapek′s solution agar）倍地
中の炭素源を米生澱粉（１％）にかえ、30℃で培養する
と、明確なハローを形成する。Czapek's solution agar The carbon source in the medium is changed to rice starch (1%) and cultivated at 30 ° C to form a clear halo.
（２）生理的性質 澱粉の加水分解性：陽性 酵素に対する態度：好気性 生育のpH範囲:pH3.5〜12 生育の温度範囲:15〜35℃ これらの観察結果より、本菌は不完全菌のペニシリウム
属に属する。ペニシリは複輪生体〜非対称体であり、分
生子柄は通常長く、コロニーは羊毛状、白色から後に灰
緑色となることにより、ペニシリウム・コマネ（P.comm
ane）系に含まれる。(2) Physiological properties Hydrolyzability of starch: Positive Attitude toward enzymes: Aerobic Growth pH range: pH 3.5-12 Growth temperature range: 15-35 ℃ From these observations, this bacterium is an incomplete bacterium. Of the genus Penicillium. Penicilli are polymorphic to asymmetrical, conidia stalks are usually long, colonies become wooly, white to grayish green, and thus Penicillium comemane (P. comm.
ane) system.
この系の中には８種含まれているが、麦芽寒天倍地上で
よく胞子形成すること、分生子の表面が細かな粗面であ
ることなどからペニシリウム・ラノスム・ウエストリン
グ（Penicillium lanosum Westling）と同定され、その
代表的なペニシリウム・ラノスムOGR−１株を、昭和60
年10月23日に工業技術院微生物工業技術研究所に微工研
菌寄第8491号（FERM Ｐ−8491）として寄託した。Eight kinds are contained in this system, but due to the fact that sporulation occurs well on malt agar and that the surface of conidia is fine and rough, Penicillium lanosum Westling The representative Penicillium lanosum OGR-1 strain was identified as
On October 23, 2013, it was deposited in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 8491 (FERM P-8491).
本菌は次の如く分離、純化される。分離は試料の適量を
シャーレ内にとり、その上より肉汁寒天Buillon aga
r）培地中の炭素源を米生澱粉（１％）に改変した培地
を流し込むことにより行なった。30℃で３日間インキュ
ベートした後、コロニーの周辺に米生澱粉が溶解してい
る明確なハローと呼ばれる領域が形成されたものを採取
し、前記と同様な培地で培養し、ハローの形成が再確認
されたものを分離、保存する（１次パス菌株と称す
る）。This bacterium is separated and purified as follows. For separation, take an appropriate amount of sample in a Petri dish and place it on the broth agar Buillon aga.
r) It was carried out by pouring a medium in which the carbon source in the medium was changed to rice starch (1%). After incubating at 30 ° C for 3 days, a colony around which a clear rice solute-dissolved area called a halo was formed was collected and cultured in a medium similar to the above to re-establish the halo formation. The confirmed one is separated and stored (referred to as the first-pass strain).
さらに、１次パス菌株について、肉汁培地中の炭素源を
米生澱粉（１％）で改変した液体培地中で30℃、５〜７
日間振盪培養し、その培地ブロスを粗酵素液として生澱
粉分解活性および可溶性澱粉分解活性を測定し、可溶性
澱粉の分解速度に対する生澱粉分解速度の比（R/S比）
が20以上のもの６株を選択し、６株の中で最もR/S比の
大きな株としてOGR−１を選択した。R/S比は下記のよう
な式で与えられる。Furthermore, for the first-pass strain, the carbon source in the broth medium was modified with rice starch (1%) in a liquid medium at 30 ° C for 5 to 7
After culturing with shaking for a day, the raw broth was used as a crude enzyme solution to measure the raw starch degrading activity and the soluble starch degrading activity, and the ratio of the raw starch degrading rate to the soluble starch degrading rate (R / S ratio)
6 strains having a ≥20 were selected, and OGR-1 was selected as the strain having the largest R / S ratio among the 6 strains. The R / S ratio is given by the following formula.
本発明により生澱粉分解酵素は前記糸状菌を栄養源培地
に接種し、培養せしめることにより製造される。培養に
用いられた培地としては、酵素誘導基質としてのエピク
ロルヒドリン架橋澱粉と、当該糸状菌が利用する栄養源
を含むものであれば何れでもよいが、本発明者らは酵素
生産用培地として基本棒地20種類、炭素源８種類、窒素
源８種類、誘導基質６種類について鋭意検討の結果、望
ましくは炭素源として１％ソルビトール、窒素源として
１％KNO3、無機成分として0.5％KH2PO4、0.01％FeCl3、
0.25％MgSO4・7H2Oを含み、誘導基質として0.5％エピク
ロルヒドリン架橋澱粉からなるpH6.0の独自の培地を用
いることが最適であることを見出した。 According to the present invention, the raw starch degrading enzyme is produced by inoculating the nutrient medium with the filamentous fungus and culturing it. The medium used for the culture may be any one as long as it contains epichlorohydrin-crosslinked starch as an enzyme-inducing substrate and a nutrient source utilized by the filamentous fungus, but the present inventors use it as a basic medium for enzyme production. As a result of diligent studies on 20 types of ground, 8 types of carbon sources, 8 types of nitrogen sources, and 6 types of derived substrates, it is desirable that 1% sorbitol is used as a carbon source, 1% KNO 3 is used as a nitrogen source, and 0.5% KH 2 PO 4 is used as an inorganic component. , 0.01% FeCl 3 ,
Comprises 0.25% MgSO 4 · 7H 2 O , using the unique media pH6.0 consisting of 0.5% epichlorohydrin crosslinked starch as an inducer substrate was found to be optimal.
培養法としては通気培養法が好適である。条件としては
1vvm30℃付近で行なわれる。２〜３日培養後、培養液は
次の操作に付される。生澱粉分解酵素は通常、培養濾液
中に存在するので、濾過または遠心分離等の手段を用い
ることにより、培養物から菌体を分離した後、80％硫安
による塩析を行なう。次に塩析により析出したタンパク
の沈澱を遠心分離により集め、蒸留水に溶解後、透析、
内液を80％アセトン沈澱処理し、さらに透析を行ない、
凍結乾燥し、粗酵素粉末を得る。この粗酵素は、次のよ
うな理化学的性質を有する。The aeration culture method is preferable as the culture method. As a condition
1vvm is performed around 30 ℃. After culturing for 2 to 3 days, the culture solution is subjected to the following operation. Since the raw starch degrading enzyme is usually present in the culture filtrate, the cells are separated from the culture by using a means such as filtration or centrifugation, and then salting out with 80% ammonium sulfate is performed. Next, the protein precipitates deposited by salting out were collected by centrifugation, dissolved in distilled water, dialyzed,
The inner solution is treated with 80% acetone precipitation, and then dialyzed.
Lyophilize to obtain crude enzyme powder. This crude enzyme has the following physicochemical properties.
（１）作用：単独使用または市販のα−アミラーゼ（ア
ミラーゼＫ）と共に作用して生（無蒸煮）の澱粉を加水
分解しグルコースを得る。（第１図および第２図参照） 第１図は、各々、生の米澱粉、小麦澱粉、トウモロコシ
澱粉、甘薯澱粉および馬鈴薯澱粉の水分散液に該新規酵
素を200μｇタンパク当量でpH5にて作用させ、37℃でイ
ンキュベートした際のインキュベート時間と加水分解率
の関係を示すグラフである。また、第２図は、該酵素約
２単位と糊化澱粉分解活性を0.1Uに調整した市販のα−
アミラーゼの混合物を同様に米澱粉に作用させた場合の
グラフである。なお、活性はμmol−G1c/分で表し、加
水分解率はｇ−生成G1c/g−澱粉で算出した。(1) Action: Used alone or in combination with a commercially available α-amylase (amylase K) to hydrolyze raw (non-steamed) starch to obtain glucose. (See FIG. 1 and FIG. 2) FIG. 1 shows the action of the novel enzyme in an aqueous dispersion of raw rice starch, wheat starch, corn starch, sweet potato starch and potato starch at 200 μg protein equivalent at pH 5. 3 is a graph showing the relationship between the incubation time and the hydrolysis rate when allowed to incubate at 37 ° C. Further, Fig. 2 shows that about 2 units of the enzyme and a commercially available α-in which the degrading activity of gelatinized starch was adjusted to 0.1U.
It is a graph when a mixture of amylase is similarly made to act on rice starch. The activity was expressed in μmol-G1c / min, and the hydrolysis rate was calculated in g-produced G1c / g-starch.
（２）至適pH:pH5.0（第３図参照） 第３図には各pHの緩衝液中での活性を示す。(2) Optimum pH: pH 5.0 (see FIG. 3) FIG. 3 shows the activity in the buffer solution at each pH.
（３）pH安定生：各pHの緩衝液中で20時間処理した場
合、pH3〜７において90％以上の残存活性を示す。（第
４図参照） （４）温度安定性：各温度で10分間処理した場合、50℃
まで安定。（第５図参照） 残存活性は当初の活性を100％とした場合の残存する活
性の割合である。(3) pH stable production: When treated in a buffer solution of each pH for 20 hours, 90% or more of residual activity is shown at pH 3 to 7. (See Fig. 4) (4) Temperature stability: 50 ℃ when treated at each temperature for 10 minutes.
Stable up to. (See FIG. 5) The residual activity is the ratio of the remaining activity when the initial activity is 100%.
このようにして得られた生澱粉分解酵素は直接そのま
ま、あるいは市販のα−アミラーゼ剤を常法により適宜
配合してなる酵素剤として、蒸煮工程なしに生の澱粉を
加水分解するために、通常の酵素剤と同様に使用でき、
これにより醸造工程の省力化、省エネルギー化、さらに
は農産物の未利用澱粉のエネルギー的回収に利用できる
ものである。The raw starch degrading enzyme thus obtained is directly used as it is, or as an enzyme agent appropriately blended with a commercially available α-amylase agent by a conventional method, in order to hydrolyze raw starch without a cooking step, it is usually used. It can be used in the same way as
This can be used for labor saving and energy saving in the brewing process, and also for energy recovery of unused starch of agricultural products.
次に実施例を挙げて説明する。Next, examples will be described.
実施例１ 酵素生産のための培養 ペニシリウム・ラノスム（P.lanosum）OGR−１の保存
スラントより、ブイヨン（pH6.0）の平板培地上に一白
金耳接種し、30℃、２日間培養する。Example 1 Culture for Enzyme Production One platinum loop was inoculated from a preserved slant of Penicillium lanosum OGR-1 on a plate medium of broth (pH 6.0) and cultured at 30 ° C. for 2 days.
培養後、形成された本菌のコロニー上に殺菌水10mlを
滴下し、菌体懸濁液を作り、殺菌済みガラスフィルター
（3G−３）を用い、濾過し、胞子懸濁液を得る。After culturing, 10 ml of sterilized water was dropped on the formed colonies of the present bacterium to prepare a microbial cell suspension, which was filtered using a sterilized glass filter (3G-3) to obtain a spore suspension.
次に胞子濃度を１×106個/ml〜１×107個/ml、望まし
くは５×106＋個/mlに調整し、これを１％エピクロルヒ
ドリン架橋澱粉を含むブイヨン培地（pH6.0）100mlにつ
き1ml接種し、30℃、３日間程度振盪培養する。（前培
養）。Then, the spore concentration was adjusted to 1 × 10 6 cells / ml to 1 × 10 7 cells / ml, preferably 5 × 10 6 cells / ml, and this was adjusted to a broth medium containing 1% epichlorohydrin-crosslinked starch (pH 6.0). ) Inoculate 1 ml per 100 ml and culture with shaking at 30 ° C for about 3 days. (Preculture).
にて得られた前培養培地を種とし、１％のソルビト
ール、１％KNO3、0.5％KH2PO4、0.01％FeCl3、0.25％Mg
SO47H2O、0.5％エピクロルヒドリン架橋澱粉からなるpH
6.0の本培養培地に20％の割合になるよう添加し、30
℃、1vvmの条件下で通気培養を行なう。The pre-culture medium obtained in 1. was used as a seed, 1% sorbitol, 1% KNO 3 , 0.5% KH 2 PO 4 , 0.01% FeCl 3 , 0.25% Mg
SO 4 7H 2 O, pH consisting of 0.5% epichlorohydrin crosslinked starch
Add to the main culture medium of 6.0 at a ratio of 20%,
Aeration culture is performed under the condition of 1 vvm at ℃.
実施例２ 粗酵素粉末の調製 培養終了後、濾過または遠心分離により菌体を除去
後、培養濾液を得る。Example 2 Preparation of crude enzyme powder After completion of the culture, the bacterial cells are removed by filtration or centrifugation to obtain a culture filtrate.
培養濾液に対し、望ましくは０℃に冷却下ないし５℃
以下の条件でスターラーで静かに撹拌しながら、培養瀘
液に80％飽和になるように固形硫安を徐々に加え塩所を
行なった。The culture filtrate is preferably cooled to 0 ° C to 5 ° C
While gently stirring with a stirrer under the following conditions, solid ammonium sulfate was gradually added to the culture filtrate to 80% saturation to carry out salting.
塩所により析出したタンパクの沈澱は、1000Gで５分
間遠心分離し集め、これを蒸留水に溶解後、５℃で24時
間透析を行なった。The protein precipitate deposited at the salt place was collected by centrifugation at 1000 G for 5 minutes, dissolved in distilled water, and dialyzed at 5 ° C. for 24 hours.
この透析液に対し、５℃以下で80％アセトン処理を行
ない、再度1000G、５分間遠心分離することにより沈澱
物を得、蒸留水に溶解後、と同条件で透析を行なっ
た。This dialysate was subjected to 80% acetone treatment at 5 ° C. or lower, centrifuged again at 1000 G for 5 minutes to obtain a precipitate, dissolved in distilled water, and then dialyzed under the same conditions.
透析液を凍結乾燥することにより粗酵素粉末を得た。
該酵素粉末は前記のような理化学的性質を有していた。Crude enzyme powder was obtained by freeze-drying the dialysate.
The enzyme powder had the physicochemical properties as described above.
第１図および第２図は本発明で得られた生澱粉分解酵素
の加水分解活性を示すグラフ、第３図は該酵素の至適pH
を、第４図はpH安定性を、また、第５図は温度安定性を
示すグラフである。1 and 2 are graphs showing the hydrolysis activity of the raw starch degrading enzyme obtained in the present invention, and FIG. 3 is the optimum pH of the enzyme.
FIG. 4 is a graph showing pH stability, and FIG. 5 is a graph showing temperature stability.
Claims (2)
産菌を、エピクロルヒドリン架橋澱粉、ソルビトール、
KNO3、K2HPO4、FeCl3およびMgSO4から成る倍地で液体培
養し、その培養瀘液より生澱粉分解酵素を得ることを特
徴とする下記の理化学的性質を有する生澱粉分解酵素の
製造法。 作用：α−アミラーゼと共に作用して生（無蒸煮）の
澱粉を加水分解しグルコースを得る。 至適pH:pH5.0 pH安定生：各pHの緩衝液中で20時間処理した場合、pH
3〜７において90％以上の残存活性を示す。 温度安定性：各温度で10分間処理した場合、50℃まで
安定。1. A raw starch-degrading enzyme-producing bacterium belonging to the genus Penicillium is treated with epichlorohydrin-crosslinked starch, sorbitol,
A raw starch degrading enzyme having the following physicochemical properties, which is characterized in that the raw starch degrading enzyme is obtained by liquid culture in a medium composed of KNO 3 , K 2 HPO 4 , FeCl 3 and MgSO 4, and obtaining the raw starch degrading enzyme from the culture filtrate. Manufacturing method. Action: Acts together with α-amylase to hydrolyze raw (non-steamed) starch to obtain glucose. Optimum pH: pH 5.0 pH stability: pH when treated in a buffer solution of each pH for 20 hours
In 3 to 7, 90% or more of residual activity is shown. Temperature stability: Stable up to 50 ° C when treated at each temperature for 10 minutes.
ノスム（Penicillium lanosum）OGR−１である特許請求
の範囲第（１）項記載の生澱粉分解酵素の製造法。2. The method for producing a raw starch degrading enzyme according to claim 1, wherein the raw starch degrading enzyme producing bacterium is Penicillium lanosum OGR-1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61202873A JPH0763364B2 (en) | 1986-08-28 | 1986-08-28 | Method for producing raw starch degrading enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61202873A JPH0763364B2 (en) | 1986-08-28 | 1986-08-28 | Method for producing raw starch degrading enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6359887A JPS6359887A (en) | 1988-03-15 |
| JPH0763364B2 true JPH0763364B2 (en) | 1995-07-12 |
Family
ID=16464609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61202873A Expired - Lifetime JPH0763364B2 (en) | 1986-08-28 | 1986-08-28 | Method for producing raw starch degrading enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0763364B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5188956A (en) * | 1988-07-01 | 1993-02-23 | Showa Denka K.K. | Thermostable amylase |
| GB0129864D0 (en) * | 2001-12-13 | 2002-02-06 | Danisco | Animal feed |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3922196A (en) * | 1974-01-28 | 1975-11-25 | Cpc International Inc | Enzymatic hydrolysis of granular starch |
| JPS5718991A (en) * | 1980-07-10 | 1982-01-30 | Ueda Kagaku Kogyo Kk | Liquefaction and saccharification of raw starch substance without steaming or boiling |
| JPS59140896A (en) * | 1983-01-17 | 1984-08-13 | Norin Suisansyo Shokuhin Sogo Kenkyusho | Saccharification of starch using enzyme produced by fungus of chalara genus |
| JPS59159779A (en) * | 1983-03-02 | 1984-09-10 | Tax Adm Agency | Preparation of glucoamylase by schizophyllum commune fr. |
-
1986
- 1986-08-28 JP JP61202873A patent/JPH0763364B2/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3922196A (en) * | 1974-01-28 | 1975-11-25 | Cpc International Inc | Enzymatic hydrolysis of granular starch |
| JPS5718991A (en) * | 1980-07-10 | 1982-01-30 | Ueda Kagaku Kogyo Kk | Liquefaction and saccharification of raw starch substance without steaming or boiling |
| JPS59140896A (en) * | 1983-01-17 | 1984-08-13 | Norin Suisansyo Shokuhin Sogo Kenkyusho | Saccharification of starch using enzyme produced by fungus of chalara genus |
| JPS59159779A (en) * | 1983-03-02 | 1984-09-10 | Tax Adm Agency | Preparation of glucoamylase by schizophyllum commune fr. |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6359887A (en) | 1988-03-15 |
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