JP3747083B2 - Novel amylase, its production and use - Google Patents

Description

【０００９】
有性胞子が観察されないため不完全菌類として分類され、分生子殻、分生子層を形成しないので不完全糸状菌綱（Hyphomycetes）に属する。更に、菌糸が無色で細い、分生子柄が分岐しない、基部から先端に向かい徐々に細くなる、分生子が単細胞、無色、分生子塊になることなどからアクレモニウム（Acremonium）属に分類され、アクレモニウム・エスピー YT-78と命名した。
【００１０】
参考文献
▲１▼ Domsch, K. H., Gams, W., and Anderson, T. -H. 1980.
"Comopendium of soil fungi, Vol. 1" p.16-29,
Academic Press, London.
▲２▼ Tokumasu, S. 1991.
Genus Acremonium. J. Antibact. Antifung. Agent. 19巻, p.47-53
【００１１】
尚、本菌株は、通商産業省工業技術院生命工学工業技術研究所に第15097号（FERM P-15097）として寄託されている。
【００１２】
本菌株を使用して新規なアミラーゼを生産蓄積させる為の培養方法としては、液体培養法、固体培養法の何れでもよいが、好ましくは液体培養法が行われる。具体的に培養法について述べると、液体培養培地の場合は、当該微生物が良好に生育し、酵素を順調に生産するために必要な炭素源、窒素源、無機塩、必要な栄養源等を含有する合成培地又は天然培地があげられる。
【００１３】
例えば、炭素源としては、澱粉又はその組成画分、焙焼デキストリン、加工澱粉、澱粉誘導体、物理処理澱粉及びα−澱粉等の炭水化物が使用できる。具体例としては、可溶性澱粉、トウモロコシ澱粉、馬鈴薯澱粉、甘藷澱粉、デキストリン、アミロペクチン、アミロース等があげられる。
【００１４】
窒素源としては、ポリペプトン、カゼイン、肉エキス、酵母エキス、コーンスティープリカー或いは大豆又は大豆粕などの抽出物等の有機窒素源物質、硫酸アンモニウム、リン酸アンモニウム等の無機塩窒素化合物、グルタミン酸等のアミノ酸類が挙げられる。
【００１５】
そして無機塩類としては、塩化第一鉄、塩化第二鉄、硫酸第一鉄、硫酸第二鉄等の鉄イオン含有化合物、リン酸カリウム塩、リン酸ナトリウム塩等のリン酸塩、硫酸マグネシウム等のマグネシウム塩、塩化カルシウム等のカルシウム塩、炭酸ナトリウム等のナトリウム塩等が用いられる。
【００１６】
培養は、振盪培養若しくは、通気攪拌培養等の好気的条件下に於いて、培地をpH４〜８の範囲、好ましくはpH６〜７の範囲に調整し、温度20〜35℃の範囲、好ましくは、25〜30℃で実施し、通常１〜５日間培養するのが望ましいが、この条件以外であっても微生物が生育し、目的とする酵素を生成する条件であれば特に制限されない。
【００１７】
ついで、これらの粗酵素液を限外ろ過膜で脱塩、濃縮した後、硫安塩析又は有機溶媒沈降等により酵素を回収する。その後、各種クロマトグラフィー、ゲルろ過等を組み合わせて高純度の新規なアミラーゼ標品を得ることができる。このようにして得られた新規なアミラーゼの酵素化学的性質について以下に述べる。
【００１８】
▲１▼ 活性測定法：１％プルラン0.5mlに0.1Mマッキルバイン緩衝液（pH5.0）0.4ml、酵素液0.1mlを加え、50℃、３０分間反応する。反応後、ソモギー試薬0.4mlを加え、20分間加熱（沸騰水中）する。冷後、ネルソン試薬0.2mlを加え、室温で20分間放置後、精製水4.0mlを加え、500nmで吸光度を測定する。１分間当たり、グルコース１μmolに相当する還元糖を生成する酵素量を１単位とする。
【００１９】
▲２▼ 基質特異性：各種基質を使用して活性測定法に準じて測定した。溶性澱粉を基質としたときの各種基質に対する活性を表１にまとめて表示する。
【００２０】
【表１】

【００２１】
表より明らかなように、0.5％基質の場合には各種澱粉、グリコーゲン、アミロペクチン、アミロース、マルトヘプタオース、マルトヘキサオース、マルトペンタオースに良く作用し、プルランにも作用するが、マルトテトラオース、マルトトリオース、マルトース、イソマルトース、イソマルトトリオース、デキストラン、各種サイクロデキストリンには殆ど作用しない。また、５％基質の場合には0.5％基質ではほとんど作用しなかったマルトテトラオース、マルトトリオース、マルトースにも作用し、イソマルトトリオースにも僅かに作用する。
本酵素について、他のアミラーゼと各種基質に対する反応性を比較する。その結果を表２に示す。対象としては、グルコアミラーゼ（Rhizopus niveus由来、Aspergillus niger由来）、α−アミラーゼ（Bacillus subtilis由来）、プルラナーゼ（Enterobacter aerogenes由来）を使用した。
【００２２】
【表２】

【００２３】
表からも明らかなように、本発明のアミラーゼはグルコアミラーゼやα−アミラーゼの基質となる溶性澱粉に作用し、更にプルラナーゼの基質となるプルランにも良く作用する広い基質特異性を有していることが特徴である。
【００２４】
▲３▼ 至適pH ：１％プルランを基質として、各種pHの緩衝液（0.1M マッキルバイン緩衝液）を用いて50℃、30分間反応した。その結果、至適pHは約5.0付近にある。（図１）
【００２５】
▲４▼ 至適温度 ：１％プルランを基質として、0.1M マッキルバイン緩衝液（pH5.0）を用いて各温度で30分間反応した。その結果、至適温度は約50℃付近にある。（図２）
【００２６】
▲５▼ pH安定性 ：１％プルランを基質として、酵素液を0.1M マッキルバイン緩衝液（pH３〜７）、0.1Mトリス−塩酸緩衝液（pH７〜８）を用いて40℃、30分間処理した後反応した。その結果、pH安定性はpH５〜９付近にある。（図３）
【００２７】
▲６▼ 熱安定性 ：１％プルランを基質として、酵素を0.1M マッキルバイン緩衝液（pH5.0）に溶解し、各温度で30分間処理した後反応した。その結果、熱安定性は40℃まで安定である。（図４）
【００２８】

【００２９】
▲８▼ 等電点 ：約5.10
【００３０】
▲９▼ 阻害剤 ：各種阻害剤（1mM又は10mM）を含む１％プルランを基質として活性測定法に従って反応した。その結果を表２に示す。
【００３１】
【表３】

【００３２】
表より明らかなようにNa⁺,K⁺,Mn²⁺,Al³⁺,Fe²⁺,Mg²⁺,Zn²⁺,Ba²⁺,Cd²⁺,Cu²⁺,Ag⁺,Ni²⁺,Co²⁺,Ca²⁺,EDTA,Ｎ−エチルマレイミド,モノヨード酢酸で実質的に阻害されず、SDS,Hg²⁺で強く阻害され、Sn²⁺,Fe³⁺,pCMB,SDSでも阻害される。
【００３３】
更に、本発明の新規なアミラーゼの用途について記載する。本発明の酵素は、上述したようにα-1,4-結合を分解する作用とα-1,6-結合を分解する作用の何れの作用も合わせ持つため、各種の食品に応用する際に有用である。例えば、清酒醸造に於ける醗酵促進、製パンにおける酵素の利用、各種果汁の清澄化にペクチナーゼと併用する等、広く利用することができる。これらの場合の利用方法については、通常の方法が適用できる。
【００３４】
以下、実施例により本発明を詳述する。なお、本発明はこれらに限定されるものではない。
【００３５】
【実施例】
実施例１ アクレモニウム・エスピー YT-78の培養
下記の組成の種培地80ml（pH6.0）を用いてアクレモニウム・エスピー YT-78（FERM P-15097）を接種し、25℃で３日間振盪培養した。
【００３６】

【００３７】
更に、上記組成の培地に0.5％プルランを追加した培地（20Ｌ）を用いて、ジャーファーメンター（30Ｌ）を使用し、25℃、250rpm、0.5vvm、64時間の条件で培養した。培養液を濾過して、粗酵素液を得た。
【００３８】
実施例２ 酵素の精製
実施例１で得られた粗酵素液（10Ｌ）を限外濾過膜濃縮後、80％飽和となるように固形硫安を添加、塩析した。次いで塩析沈殿物を脱塩後、10mMのリン酸緩衝液（pH7.0）に溶解し、CM−Toyopearl 650Mカラムにより不純蛋白質を吸着分離した。
【００３９】
非吸着画分をDEAE−Toyopearl 650Mカラムに流し、吸着した活性画分を食塩濃度を直線的に上げて溶出した。溶出活性画分に硫安を２Ｍ添加溶解し、同様の硫安を含む緩衝液で平衡化したButyl−Toyopearl 650Mカラムに流し、吸着した活性画分を硫安濃度を直線的に低下させて流出した。最後に、溶出活性画分はSepacryl 200Sカラムによるゲル濾過で精製し、単一の精製酵素を得た。本精製酵素を用いて、前述したとおりの酵素化学的性質を明らかにした。
【００４０】
実施例３ 清酒醸造の醗酵に及ぼす効果
500ml容ビーカーに25g相当の蒸米、140mlの酵素液（表４記載）及び1.3％KH₂PO₄、0.25%NaCl及び６％の乳酸を含む加工水１mlをとり、協会701号酵母懸濁液２mlを加えて15℃に放置した。２日後、75g相当の蒸米を仲留として加え、醗酵を継続した。遠心分離後、東洋濾紙No.5Cでろ過し、ろ液について各種分析した。その結果を表４に示す。
【００４１】
【表４】

【００４２】
グルコアミラーゼとして、Rhizopus niveus由来、Aspergillus niger由来及び本発明の酵素を使用して比較した。その結果、本発明の酵素を使用することによって、醪の醗酵速度が促進されることが明らかとなった。
【００４３】
実施例４ リンゴ果汁の清澄化
リンゴ（銘柄：サンフジ）果肉200gに精製水100mlを添加した後、ホモジナイザーで粉砕、綿布ろ過し、リンゴ果汁を得た。
【００４４】
果汁５mlにペクチナーゼ（ペクチナーゼＧ「アマノ」：天野製薬製）0.5mlと各種酵素（表５に記載）を加え、40℃で２時間処理した。処理後、遠心分離（3000rpm,５分）し、上清を精製水で５倍に希釈し、660nmでの吸光度を測定した。清澄化度を下記式で算出しその結果を表５に示す。
【００４５】
果汁清澄化度（％）＝（ＯＤ_C−ＯＤ_R）／ＯＤ_C×１００
【００４６】
【表５】

【００４７】
その結果、ペクチナーゼと本発明のアミラーゼを併用することによって果汁の清澄化に著しい効果が得られた。
【００４８】
実施例５ 製パンへの応用
小麦粉100g、食塩1.5g、ショートニング３g、イースト２g、ブロム酸カリ1.5mgと適当量の水を用いてストレート・ドウ法での最適水吸収量、最適攪拌時間を用いて28℃で２時間生地発酵した。ガス抜きや型入れは機械的に行い、焼成は218℃で24分行った。なお、しょ糖の添加量は適宜変化させて、本発明の酵素による効果を調べた。その結果、ドウ組成に本発明のアミラーゼを添加することによって、特にしょ糖の添加量が少ない場合において、本発明の酵素を使用することによって、ガス生成量及びローフ容積への効果が著しかった。
【００４９】
【発明の効果】
本発明により、広い基質特異性を有する新規なアミラーゼが提供される。本発明の新規なアミラーゼは、各種食品業界において、有効に利用される。
【図面の簡単な説明】
【図１】本発明の新規なアミラーゼの至適pHを示す図である。
【図２】本発明の新規なアミラーゼの至適温度を示す図である。
【図３】本発明の新規なアミラーゼのpH安定性を示す図である。
【符号の説明】
図中の−●−はマッキルバイン緩衝液を使用した場合であり、−○−はトリス−塩酸緩衝液を使用した場合を示す。
【図４】本発明の新規なアミラーゼの熱安定性を示す図である。[0001]
[Industrial application fields]
The present invention relates to a novel amylase and a method for producing the same. More particularly, the present invention relates to a novel α-amylase belonging to the genus Acremonium, obtained from a microorganism having an ability to produce amylase, and also having an activity of cleaving an α-1,6-glucoside bond, and a method for producing the same . The amylase of the present invention is used as a food enzyme and a pharmaceutical enzyme.
[0002]
[Prior art]
In general, amylase is used as a general term for enzymes that act on a substrate such as starch or glycogen to hydrolyze the glucoside bond, and is broadly classified into α-amylase, β-amylase, and glucoamylase. Each enzyme is widely distributed in animals, plants, microorganisms and the like.
[0003]
On the other hand, pullulanase known as a debranching enzyme is an enzyme that hydrolyzes an α-1,6-glucoside bond, and is known to be produced from various microorganisms.
Recently, an enzyme having both amylase activity and pullulanase activity as described above has been reported. That is, an enzyme derived from Bacillus subtilis [Agri. Biol. Chem., 51, 9 (1987)], an enzyme derived from Bacillus circulans [Biochim. Biophys. Acta, 991, 188 ( 1989)], an enzyme derived from Clostridium thermohydrosulfuricum (Japanese Examined Patent Publication No. 64-60376), and the like.
[0004]
[Problems to be solved by the invention]
However, these enzymes having both amylase activity and pullulanase activity are not satisfactory in their substrate specificity, and the development of a novel amylase that acts on a wider substrate has been desired.
[0005]
[Means for Solving the Problems]
In view of such circumstances, the present inventors have intensively studied, and as a result, found that microorganisms newly collected from soil produce a large amount of novel amylase having a broader substrate specificity, and completed the present invention. .
[0006]
The properties of microorganisms newly collected from soil in potato dextrose agar medium (Eiken) are described below.
[0007]
Growth state Growth degree: Colony diameter is 23-25mm (25 ° C, 7 days)
Colony surface: fluffy, white colony back side: ocher [0008]

[0009]
Since no sexual spores are observed, it is classified as an incomplete fungus, and it does not form a conidial shell or conidial layer, so it belongs to the Hyphomycetes. Furthermore, the mycelium is colorless and thin, the conidia do not branch, gradually narrows from the base to the tip, the conidia become single cells, colorless, conidial mass, etc., and is classified into the genus Acremonium (Acremonium), Named Acremonium SP YT-78.
[0010]
Reference (1) Domsch, KH, Gams, W., and Anderson, T. -H. 1980.
"Comopendium of soil fungi, Vol. 1" p.16-29,
Academic Press, London.
▲ 2 ▼ Tokumasu, S. 1991.
Genus Acremonium. J. Antibact. Antifung. Agent. Volume 19, p.47-53
[0011]
This strain has been deposited as No. 15097 (FERM P-15097) at the Institute of Biotechnology, Ministry of International Trade and Industry.
[0012]
As a culture method for producing and accumulating a novel amylase using this strain, either a liquid culture method or a solid culture method may be used, but a liquid culture method is preferably performed. Specifically, in the case of a liquid culture medium, the culture method contains a carbon source, a nitrogen source, an inorganic salt, a necessary nutrient source, etc. necessary for the microorganism to grow well and produce the enzyme smoothly. A synthetic medium or a natural medium.
[0013]
For example, as the carbon source, carbohydrates such as starch or a composition fraction thereof, roasted dextrin, modified starch, starch derivative, physically treated starch and α-starch can be used. Specific examples include soluble starch, corn starch, potato starch, sweet potato starch, dextrin, amylopectin, and amylose.
[0014]
Examples of nitrogen sources include polypeptone, casein, meat extract, yeast extract, corn steep liquor, organic nitrogen source substances such as extracts such as soybean or soybean meal, inorganic salt nitrogen compounds such as ammonium sulfate and ammonium phosphate, and amino acids such as glutamic acid. Kind.
[0015]
And as inorganic salts, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate and other iron ion-containing compounds, potassium phosphate, phosphate sodium phosphate, magnesium sulfate, etc. Magnesium salts, calcium salts such as calcium chloride, sodium salts such as sodium carbonate, and the like are used.
[0016]
The culture is performed under aerobic conditions such as shaking culture or aeration and agitation culture, and the medium is adjusted to a pH range of 4 to 8, preferably a pH range of 6 to 7, and a temperature range of 20 to 35 ° C., preferably However, it is preferably carried out at 25-30 ° C. and usually cultured for 1-5 days. However, there is no particular limitation as long as the conditions allow the microorganism to grow and produce the target enzyme even under other conditions.
[0017]
Subsequently, these crude enzyme solutions are desalted and concentrated with an ultrafiltration membrane, and then the enzyme is recovered by ammonium sulfate salting out or organic solvent precipitation. Then, a high purity new amylase preparation can be obtained by combining various types of chromatography, gel filtration and the like. The enzymochemical properties of the novel amylase thus obtained are described below.
[0018]
(1) Activity measurement method: 0.4 ml of 0.1 M mucklevine buffer (pH 5.0) and 0.1 ml of enzyme solution are added to 0.5 ml of 1% pullulan and reacted at 50 ° C. for 30 minutes. After the reaction, add 0.4 ml of somology reagent and heat for 20 minutes (boiling water). After cooling, 0.2 ml of Nelson reagent is added, and after standing at room temperature for 20 minutes, 4.0 ml of purified water is added, and the absorbance is measured at 500 nm. The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute is defined as 1 unit.
[0019]
(2) Substrate specificity: Measured according to the activity measurement method using various substrates. Table 1 summarizes the activity against various substrates when soluble starch is used as the substrate.
[0020]
[Table 1]

[0021]
As is clear from the table, in the case of 0.5% substrate, it works well on various starches, glycogen, amylopectin, amylose, maltoheptaose, maltohexaose, maltopentaose, and also acts on pullulan, but maltotetraose, Almost no effect on maltotriose, maltose, isomaltose, isomaltotriose, dextran and various cyclodextrins. In the case of 5% substrate, it also acts on maltotetraose, maltotriose and maltose which hardly acted on 0.5% substrate, and slightly acts on isomalttriose.
The reactivity of this enzyme with other amylases and various substrates is compared. The results are shown in Table 2. As subjects, glucoamylase (derived from Rhizopus niveus, derived from Aspergillus niger), α-amylase (derived from Bacillus subtilis), and pullulanase (derived from Enterobacter aerogenes) were used.
[0022]
[Table 2]

[0023]
As is apparent from the table, the amylase of the present invention has a broad substrate specificity that acts on soluble starch as a substrate for glucoamylase and α-amylase, and also acts well on pullulan as a substrate for pullulanase. It is a feature.
[0024]
(3) Optimum pH: Reaction was carried out at 50 ° C. for 30 minutes using various pH buffers (0.1 M McKilvine buffer) with 1% pullulan as a substrate. As a result, the optimum pH is around 5.0. (Figure 1)
[0025]
(4) Optimum temperature: The reaction was carried out at each temperature for 30 minutes using 0.1 M McIlvine buffer (pH 5.0) with 1% pullulan as a substrate. As a result, the optimum temperature is around 50 ° C. (Figure 2)
[0026]
(5) pH stability: Using 1% pullulan as a substrate, the enzyme solution was treated with 0.1 M mucklevine buffer (pH 3-7) and 0.1 M Tris-HCl buffer (pH 7-8) at 40 ° C. for 30 minutes. After reaction. As a result, the pH stability is around pH 5-9. (Figure 3)
[0027]
(6) Thermal stability: 1% pullulan was used as a substrate, the enzyme was dissolved in 0.1 M McKilvine buffer (pH 5.0), reacted at each temperature for 30 minutes, and then reacted. As a result, the thermal stability is stable up to 40 ° C. (Fig. 4)
[0028]

[0029]
(8) Isoelectric point: approx. 5.10
[0030]
{Circle around (9)} Inhibitor: Reaction was carried out according to an activity measurement method using 1% pullulan containing various inhibitors (1 mM or 10 mM) as a substrate. The results are shown in Table 2.
[0031]
[Table 3]

[0032]
As is clear from the table, Na ⁺ , K ⁺ , Mn ²⁺ , Al ³⁺ , Fe ²⁺ , Mg ²⁺ , Zn ²⁺ , Ba ²⁺ , Cd ²⁺ , Cu ²⁺ , Ag ⁺ , Ni ²⁺ , Co ²⁺ , Ca ²⁺ , EDTA, N-ethylmaleimide, monoiodoacetic acid, not substantially inhibited, SDS, Hg ²⁺ strongly inhibited, Sn ²⁺ , Fe ³⁺ , pCMB, SDS also inhibited The
[0033]
Furthermore, the use of the novel amylase of the present invention is described. As described above, the enzyme of the present invention has both the action of degrading α-1,4-bonds and the action of degrading α-1,6-bonds. Useful. For example, it can be widely used such as fermentation promotion in sake brewing, use of enzymes in bread making, and pectinase for clarification of various fruit juices. As for the utilization method in these cases, a normal method can be applied.
[0034]
Hereinafter, the present invention will be described in detail by way of examples. The present invention is not limited to these.
[0035]
【Example】
Example 1 Culture of Acremonium sp. YT-78 Inoculated with Acremonium sp. YT-78 (FERM P-15097) using 80 ml (pH 6.0) of seed medium having the following composition and shaken at 25 ° C. for 3 days. Cultured.
[0036]

[0037]
Furthermore, a medium (20 L) obtained by adding 0.5% pullulan to the medium having the above composition was used, and cultured using a jar fermenter (30 L) at 25 ° C., 250 rpm, 0.5 vvm, and 64 hours. The culture solution was filtered to obtain a crude enzyme solution.
[0038]
Example 2 Purification of Enzyme After the crude enzyme solution (10 L) obtained in Example 1 was concentrated by ultrafiltration membrane, solid ammonium sulfate was added and salted out to 80% saturation. Next, the salted out precipitate was desalted and then dissolved in 10 mM phosphate buffer (pH 7.0), and the impure protein was separated by adsorption using a CM-Toyopearl 650M column.
[0039]
The non-adsorbed fraction was applied to a DEAE-Toyopearl 650M column, and the adsorbed active fraction was eluted by increasing the salt concentration linearly. 2M ammonium sulfate was added and dissolved in the eluted active fraction, and it was passed through a Butyl-Toyopearl 650M column equilibrated with a buffer solution containing the same ammonium sulfate, and the adsorbed active fraction was discharged with the ammonium sulfate concentration decreased linearly. Finally, the eluted active fraction was purified by gel filtration using a Sepacryl 200S column to obtain a single purified enzyme. Using this purified enzyme, the enzyme chemistry as described above was clarified.
[0040]
Example 3 Effect of Sake Brewing on Fermentation
In a 500 ml beaker, take 25 ml of steamed rice, 140 ml of enzyme solution (described in Table 4) and 1 ml of processed water containing 1.3% KH ₂ PO ₄ , 0.25% NaCl and 6% lactic acid. And left at 15 ° C. Two days later, steamed rice equivalent to 75 g was added as a medium and the fermentation was continued. After centrifugation, it was filtered with Toyo filter paper No. 5C, and the filtrate was analyzed in various ways. The results are shown in Table 4.
[0041]
[Table 4]

[0042]
As glucoamylases, Rhizopus niveus origin, Aspergillus niger origin and the enzyme of the present invention were used for comparison. As a result, it became clear that the fermentation rate of koji was accelerated by using the enzyme of the present invention.
[0043]
Example 4 Clarification of Apple Juice After adding 100 ml of purified water to 200 g of apple (brand: Sanfuji) pulp, it was pulverized and filtered with a homogenizer to obtain apple juice.
[0044]
0.5 ml of pectinase (Pectinase G “Amano”: Amano Pharmaceutical) and various enzymes (described in Table 5) were added to 5 ml of fruit juice and treated at 40 ° C. for 2 hours. After the treatment, it was centrifuged (3000 rpm, 5 minutes), the supernatant was diluted 5 times with purified water, and the absorbance at 660 nm was measured. The clarification degree was calculated by the following formula, and the result is shown in Table 5.
[0045]
Fruit juice clarification degree (%) = (OD _C −OD _R ) / OD _C × 100
[0046]
[Table 5]

[0047]
As a result, by using pectinase and the amylase of the present invention in combination, a remarkable effect was obtained for clarification of fruit juice.
[0048]
Example 5 Application to breadmaking Using 100g of wheat flour, 1.5g of salt, 3g of shortening, 2g of yeast, 1.5mg of potassium bromate and an appropriate amount of water and using the optimum water absorption and optimum stirring time by the straight dough method. The dough was fermented at 28 ° C for 2 hours. Degassing and mold filling were performed mechanically, and firing was performed at 218 ° C. for 24 minutes. In addition, the addition amount of sucrose was changed suitably and the effect by the enzyme of this invention was investigated. As a result, by adding the amylase of the present invention to the dough composition, particularly when the amount of sucrose added is small, the use of the enzyme of the present invention has a significant effect on the amount of gas produced and the loaf volume.
[0049]
【The invention's effect】
The present invention provides a novel amylase having a wide substrate specificity. The novel amylase of the present invention is effectively used in various food industries.
[Brief description of the drawings]
FIG. 1 is a graph showing the optimum pH of the novel amylase of the present invention.
FIG. 2 is a graph showing the optimum temperature of the novel amylase of the present invention.
FIG. 3 is a graph showing the pH stability of the novel amylase of the present invention.
[Explanation of symbols]
In the figure,-●-indicates the case where the Muckilvine buffer is used, and -O- indicates the case where the Tris-HCl buffer is used.
FIG. 4 is a graph showing the thermal stability of the novel amylase of the present invention.

Claims (6)

A novel amylase derived from the genus Acremonium having the following enzymatic chemistry.
a. Action It acts on polysaccharides and oligosaccharides to release glucose.
b. Substrate specificity It acts on starch, glycogen, amylose, amylopectin, maltoheptaose, maltohexaose and maltopentaose, and also acts on pullulan, maltose and panose, but acts almost exclusively on isomaltose, dextran and cyclodextrin. do not do.
c. Optimum pH 5.0
d. Optimal temperature 50 ℃
e. pH stability pH 5-9 (40 ℃, 30 minutes)
f. Thermal stability 40 ℃, 30 minutes (pH5.0)
g. Molecular weight 74,000 (SDS-PAGE) A method for producing a novel amylase, comprising culturing a microorganism belonging to the genus Acremonium in a nutrient medium, accumulating and collecting a novel amylase having the following enzyme chemical properties.
a. Action It acts on polysaccharides and oligosaccharides to release glucose.
b. Substrate specificity It acts on starch, glycogen, amylose, amylopectin, maltoheptaose, maltohexaose and maltopentaose, and also acts on pullulan, maltose and panose, but acts almost exclusively on isomaltose, dextran and cyclodextrin. do not do.
c. Optimum pH 5.0
d. Optimal temperature 50 ℃
e. pH stability pH 5-9 (40 ° C, 30 minutes)
f. Thermal stability 40 ℃, 30 minutes (pH5.0)
g. Molecular weight 74,000 (SDS-PAGE)   A food enzyme comprising the novel amylase according to claim 1.   A method for promoting koji fermentation of sake using the novel amylase according to claim 1.   A method for clarifying fruit juice using the novel amylase and pectinase according to claim 1 in combination.   A method for producing bread obtained by preparing dough and baking using the novel amylase according to claim 1.

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