JPH02113886A - Novel alpha-amylase, its production and production of sugars using same enzyme - Google Patents
Novel alpha-amylase, its production and production of sugars using same enzymeInfo
- Publication number
- JPH02113886A JPH02113886A JP63265614A JP26561488A JPH02113886A JP H02113886 A JPH02113886 A JP H02113886A JP 63265614 A JP63265614 A JP 63265614A JP 26561488 A JP26561488 A JP 26561488A JP H02113886 A JPH02113886 A JP H02113886A
- Authority
- JP
- Japan
- Prior art keywords
- amylase
- starch
- enzyme
- production
- thermomonospora
- 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
- 108090000637 alpha-Amylases Proteins 0.000 title claims abstract description 63
- 102000004139 alpha-Amylases Human genes 0.000 title claims abstract description 61
- 229940024171 alpha-amylase Drugs 0.000 title claims abstract description 56
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 47
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 47
- 229940088598 enzyme Drugs 0.000 title claims abstract description 47
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 23
- 235000000346 sugar Nutrition 0.000 title description 18
- 150000008163 sugars Chemical class 0.000 title description 2
- 229920002472 Starch Polymers 0.000 claims abstract description 51
- 235000019698 starch Nutrition 0.000 claims abstract description 51
- 239000008107 starch Substances 0.000 claims abstract description 50
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- 238000000034 method Methods 0.000 claims abstract description 17
- 241000203640 Thermomonospora Species 0.000 claims abstract description 15
- 238000002523 gelfiltration Methods 0.000 claims abstract description 6
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229920000945 Amylopectin Polymers 0.000 claims abstract description 5
- 229920000856 Amylose Polymers 0.000 claims abstract description 5
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- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 claims abstract description 5
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 claims abstract description 5
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- 150000001720 carbohydrates Chemical class 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 5
- 239000004382 Amylase Substances 0.000 claims description 4
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- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims description 3
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- ZCLAHGAZPPEVDX-MQHGYYCBSA-N panose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@@H]1CO[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 ZCLAHGAZPPEVDX-MQHGYYCBSA-N 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 2
- 108010019077 beta-Amylase Proteins 0.000 claims description 2
- 229910052793 cadmium Inorganic materials 0.000 claims description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- 229940097362 cyclodextrins Drugs 0.000 claims description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims description 2
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- 239000011701 zinc Substances 0.000 claims description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 3
- FYGDTMLNYKFZSV-DZOUCCHMSA-N alpha-D-Glcp-(1->4)-alpha-D-Glcp-(1->4)-D-Glcp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-DZOUCCHMSA-N 0.000 abstract 1
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- 230000002378 acidificating effect Effects 0.000 description 8
- 244000005700 microbiome Species 0.000 description 7
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- 230000001580 bacterial effect Effects 0.000 description 6
- 238000011109 contamination Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
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- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Classifications
-
- 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
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、マルトース生成型の新規なα−アミラーゼ並
びにそれを用いた糖類の製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel maltose-producing α-amylase and a method for producing saccharides using the same.
更）ご詳しくは、サーモモノスポラ属に属する好熱性放
線菌を培養して得ちれる、耐熱性で、デンプンもしくは
、その部分加水分解物からマルトースを主成分とする糖
質を生成する新規なα−アミラーゼ、並びにそれを用い
た糖質の製造法に関する。(Additional) For more information, please refer to Thermomonospora genus thermophilic actinomycetes. The present invention relates to α-amylase and a method for producing carbohydrates using the same.
α−アミラーゼはデンプン、アミロース、アミロペクチ
ン、グリコーゲン及びそれらの部分加水分解物中α−１
，４−グルコピラノシド結合を任意に加水分解し、グル
コース、マルトースおよび種々のマルトオリゴ糖を生成
する酵素である。該α−アミラーゼは微生１勿、動物あ
るいはｈＨ勿中に広く分布している。α-Amylase is α-1 in starch, amylose, amylopectin, glycogen and their partial hydrolysates.
, 4-glucopyranoside bonds to produce glucose, maltose, and various maltooligosaccharides. The α-amylase is widely distributed in microorganisms, animals, and hH microorganisms.
α−アミラーゼのうち、微生物由来のα−アミラーゼは
例えばデンプンの液化や繊維の糊抜きなどに大量に用い
られている。例えば、バチルス（８ａｃ＋ ｌ Ｉｕｓ
） 寓：Ｂ、アミロリクエファシェンス（Ｂ、 ａｍ
ｙｌｏｌ’１ｑｕｅｆａｃｉｅｎｓ）、Ｂ、リシエニホ
ルミス（Ｂ、 ｌｉｃｈｅｎｉｆｏｒｍｉｓ）など〕
を生産するα−アミラーゼは、耐熱性に優れていること
かうデンプン糖の製造用原料として用いられる液化デン
プンの製造等に用いられている。これらの目的に用いら
れるα−アミラーゼはデンプンに作用させたときに液化
型に作用し、糊液粘度を急激に低下せしめる優れた作用
を有しているが、デンプンからの生成物はグルコース、
マルトース、及びマルト）ＩＪオースなどの一連のマル
トオリゴ糖であり本発明の目的とするマルトース生産の
ための糖化酵素としては使用できない。Among α-amylases, α-amylase derived from microorganisms is used in large quantities for, for example, liquefying starch and desizing textiles. For example, Bacillus (8ac+ l Ius
) Allegory: B, amyloliquefaciens (B, am
ylol'1quefaciens), B. licheniformis, etc.]
α-amylase, which produces α-amylase, has excellent heat resistance and is used in the production of liquefied starch, which is used as a raw material for the production of starch sugar. α-Amylase used for these purposes acts in a liquefied form when it acts on starch, and has an excellent effect of rapidly reducing the viscosity of starch, but the products from starch are glucose,
It is a series of malto-oligosaccharides such as maltose and malto)IJose, and cannot be used as a saccharifying enzyme for producing maltose, which is the object of the present invention.
一方、これろのα−アミラーゼ以外にいくつかの微生物
由来のα−アミラーゼをマルトースの生産に用いる試み
がなされている。例えば、ストレプトマイセス・バイグ
ロスコピカス（Ｓｔｒｅｐｔｏｍｙｃｅｓｈｙｇｒｏｓ
ｃｏｐ＋ｃｕｓ） （Ｄｉｅ ５ｔａｒｋｅ、 ２６
＠、４１３項、１９７３年）、ストレプトマイセス・プ
レコックス（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｐｒｅｃｏｘ
） （特開昭５０−５２２７７号）、サーモアクチノマ
イセス・ブルガリス（Ｔｈｅｒｍｏａｃｔｉｎｏｍｙｃ
ｅｓ ｖｕｌｕｇａｒｉｓ） ｃ、へｇｒｉｃ、
Ｂｉｏｌ。On the other hand, in addition to these α-amylases, attempts have been made to use α-amylases derived from several microorganisms for the production of maltose. For example, Streptomyces bygroscopicus
cop+cus) (Die 5tarke, 26
@, Section 413, 1973), Streptomyces precox
) (Japanese Unexamined Patent Publication No. 50-52277), Thermoactinomyces vulgaris
es vulgaris) c, hegric,
Biol.
Ｃｈｅｍ、４２巻、１６８１項、１９７８年）、バチル
ス・ステアロサーモフィルス（Ｂａｃｉｌｌｕｓ ｓｔ
ｅａｒｏ−ｔｈｅｒｍｏｐｈｉｌｖｓ） （Ｓｔａｒ
ｃｈ／５ｔａｒｋｅ、 ３５巻、４０５項、１９８４
年）などが報告されている。Chem, Vol. 42, Section 1681, 1978), Bacillus stearothermophilus (Bacillus st.
aero-thermophils) (Star
ch/5tarke, Volume 35, Section 405, 1984
), etc. have been reported.
しかしながらこれらのα−アミラーゼについては耐熱性
及び酵素の生産性の点で必ずしも有利なものではなかっ
た。However, these α-amylases were not necessarily advantageous in terms of heat resistance and enzyme productivity.
一方、一般にデンプン糖の生産は、５０℃以上の高温で
かつｐＨ４，５〜６．０の弱酸性条件下で行なわれてい
る。ところが、６０℃以下の反応温度では反応槽中の微
生物汚染により反応ｐＨが低下する。On the other hand, starch sugar production is generally carried out at a high temperature of 50° C. or higher and under slightly acidic conditions of pH 4.5 to 6.0. However, at a reaction temperature of 60° C. or lower, the reaction pH decreases due to microbial contamination in the reaction tank.
そこで該汚染によるｐＨ低下を補償するために、水酸化
カルシウムなどのアルカリ試薬をｐＨ調整の目的で反応
液に多量に添加しなければならなかった。Therefore, in order to compensate for the pH decrease due to the contamination, a large amount of an alkaline reagent such as calcium hydroxide must be added to the reaction solution for the purpose of pH adjustment.
このため、生産物の脱塩、精製等の後処理には多大の費
用が必要となる。従って、このような微生物汚染を防止
するためには６０℃以上に酵素反応の最適温度を有し、
同時にｐＨ低下を調整するため■アルカリ剤の添加量を
低減するために反応の至適ｐＨを中性〜弱酸性に有する
酵素が求められている。Therefore, post-processing such as desalination and purification of the product requires a large amount of cost. Therefore, in order to prevent such microbial contamination, the optimum temperature for enzyme reaction is 60°C or higher.
At the same time, in order to adjust the pH drop, (1) to reduce the amount of alkaline agent added, an enzyme having an optimum reaction pH of neutral to weakly acidic is required.
一方、バチルス・ステアロサーモフィルス（Ｂ。On the other hand, Bacillus stearothermophilus (B.
５ｔｅａｒｏｔｈｅｒ＋ｎｏｐｈｉｌｕｓ）や、サーモ
アクチノマイセス・ブルガリス（Ｔｈｅｒｍｏａｃｔｉ
ｎｏｍｙｃｅｓ ｖｕｌｕｇａｒｉｓ）のα−アミラー
ゼは、６５〜７０℃に至適温度を有しており、工業的意
味において優れた特性を有していると考えられるが、酵
素の生産性や培養の因難さのために実用的とは言えなか
った。5tearother + nophilus) and Thermoactinomyces vulgaris (Thermoacti
The α-amylase of S. nomyces vulgaris has an optimum temperature of 65 to 70°C and is considered to have excellent properties in an industrial sense, but there are problems with enzyme productivity and culture. Therefore, it could not be said to be practical.
このような状況下で高い（６０℃以上）酵素反応の至適
作用温度を有し、しかも弱酸性〜弱アルカリ性領域に安
定ｐＨ範囲を有するα−アミラーゼの開発が強く要求さ
れる。また、このような酵素を開発することによって、
デンプンからのマルトースの製造を安価かつ高い生産性
で実施することが可能となる。Under these circumstances, there is a strong demand for the development of α-amylase that has a high (60° C. or higher) optimal temperature for enzymatic reaction and a stable pH range in the weakly acidic to weakly alkaline range. In addition, by developing such enzymes,
It becomes possible to produce maltose from starch at low cost and with high productivity.
従って、本発明の第１の目的は上記要件を満足する新規
なα−アミラーゼを提供することである。Therefore, the first object of the present invention is to provide a novel α-amylase that satisfies the above requirements.
本発明の第２の目的は、上記新規α−アミラーゼを簡単
且つ高収率で製造する方法を提供することである。A second object of the present invention is to provide a method for producing the above-mentioned novel α-amylase simply and with high yield.
本発明の第３の目的は上記要件を満足する新規なα−ア
ミラーゼを使用し、デンプンもしくはその分解生成物か
ら収率よく糖類を得る方法を提供することである。A third object of the present invention is to provide a method for obtaining saccharides in good yield from starch or its decomposition products using a novel α-amylase that satisfies the above requirements.
そこで、本発明者らは培養が容易であり、酵素反応の至
適温度が６０℃以上にあり、かつ弱酸性側に至適ｐＨを
有する、温度安定性に曖れたα−アミラーゼを生産する
微生物を得るべく鋭意検索した結果、山梨県身延山中の
土壌中から採取された好熱性放線菌が上記目的を達成す
る上で極めて有用であり、これを好気的に培養すること
により、培養物中に上記要件を満足する新規α−アミラ
ーゼＴＦ−３５を高収率で生成蓄積することを見いだし
、本発明を完成したものである。Therefore, the present inventors have attempted to produce α-amylase, which is easy to culture, has an optimal temperature for enzymatic reaction of 60°C or higher, and has an optimal pH on the weakly acidic side, and which has ambiguous temperature stability. As a result of intensive searches to obtain microorganisms, thermophilic actinomycetes collected from the soil in the Minobu Mountains of Yamanashi Prefecture were found to be extremely useful in achieving the above objective. The present invention has been completed based on the discovery that a novel α-amylase TF-35 that satisfies the above requirements can be produced and accumulated in a high yield.
本発明の方法において使用する新規耐熱性α−アミラー
ゼ生産菌株は、本発明者等により新たに自然界から検索
、単離されたものであるっこの微生物の菌学的性質は下
記に示す通りである。The novel thermostable α-amylase producing strain used in the method of the present invention was newly searched for and isolated from nature by the present inventors. The mycological properties of this microorganism are as shown below. .
（１）形態的特徴 栄養寒天上での形態を以下に示す。(1) Morphological characteristics The morphology on nutrient agar is shown below.
■胞子形成菌糸の分肢法 単純分肢
■抱子形成菌糸の形態 屈曲状（ｆｌｅｘｏｕｓ）
■胞子の数 １ 個
■砲子の平面構造 平 滑
■胞子の大きさ ０．８〜０．９ Ｘｏ、
９〜１．０μ■鞭毛胞子の有無 認められない
■胞子のうの有ｍ ６１２められない■胞子
網の着生位買 気菌糸上
（２）各種培地上の性状
■栄養寒天培地
旺盛な発育で、表面は白色、裏面は淡い褐色、可溶性色
素は生成しない。又、３０℃の培養で胞子の着生がない
場合気菌糸は、暗緑色である。■Divided method for spore-forming hyphae Simple limb form ■Morphology of spore-forming hyphae Flexous
■Number of spores: 1 ■Planar structure of artillery: Smooth ■Size of spores: 0.8 to 0.9 Xo,
9-1.0 μ■ Presence or absence of flagellar spores Not observed ■ Presence of sporangia 612 Not observed ■ Epiphytic position of spore network On aerial hyphae (2) Properties on various media ■ Vigorous growth on nutrient agar medium The surface is white, the reverse side is light brown, and no soluble pigment is produced. Furthermore, when there is no spore attachment during cultivation at 30°C, the aerial mycelium is dark green.
■ペプトン・イースト・鉄寒天培地
旺盛な発育で、表面は白色、裏面は褐色、可溶性色票は
生成し：′；−・）：
■イースト・麦芽寒天培地
旺盛な発育で表面：ま薄い褐色、裏面は褐色、可溶性色
素は生成しない。■Peptone yeast/iron agar medium With vigorous growth, the surface is white, the back side is brown, and soluble color spots are produced: ´;-・): ■Yeast/malt agar medium With vigorous growth, the surface is pale brown, The underside is brown and no soluble pigment is produced.
■オートミール寒天培地
発育は旺盛ではなく、表面は白色、裏面は淡い褐色、可
溶性色素は生成しないニ
ゲルコース・アスパラギン寒天、スターチ無機塩寒天、
グリセリン・アスパラギン寒天、シュークロース・硝酸
塩寒天、チロシン寒天、ブリドハム・ボッ）ＩＪ−ブ寒
天培地上では生育しない。■Oatmeal agar medium Growth is not vigorous, the surface is white, the back side is light brown, nigercose/asparagine agar, starch inorganic salt agar, which does not produce soluble pigments,
It does not grow on glycerin/asparagine agar, sucrose/nitrate agar, tyrosine agar, Bridham Bot) IJ-B agar medium.
（３）生理的性質
■生育温度範囲 ２８〜６０℃生育最適温度
範囲 ４５〜５０℃■ゼラチンの液化
陽 性
■デンプンの加水分解 陽 性
（スターチ・イースト寒天培地上）
■脱脂牛乳の凝固 陽 性
〃 ペプトン化 陽 性
■メラニン色素の生成 陰 性
以上の菌学的（！Ｘ質についてバージエイス・マニュア
ル・オン・デタミネーティブ・バクテリオロジ−（Ｂｅ
ｒｇｅｙ’ｓ ！、４ａｎｎｕａｌ ｏｆ Ｄｅｔｅｒ
ｍｉｎａｔｉｖｅＢａｃｔｅｒｉｏｌｏｇｙ＞の第８版
を参照し本園をサーモモノスポラ・ヒ゛リゾイス（丁ｈ
ｅｒｍｏｍｏｎｏｓｐｏｒａ ｖｉｒｉｃｌｉｓ）の一
種と同定した。しかしながら、バージエイス・マニュア
ル・オン・システマテインク・バクテリオロジ−（Ｂｅ
ｒｇｅｙ’ｓ Ｍａｎｎｕａｌ ｏｆ Ｓｙｓｔｅｍａ
ｔｉｃＢａｃｔｅｒｉｏｌｏｇｙ）第１巻ではサーモモ
ノスポラ・ビリディスの記載はなくサーモモノスポラ・
ビリディス（Ｓａｃｃｈａｒｏｍｏｎｏｓｐｏｒａ ｖ
ｉｒｉｄｉｓ）と興名が変更されていだが、その菌学的
諸性質に関する詳細な記載がなされていないことからバ
ージェイス・マニュアル・オン・デタミ木−テイブ・バ
タテリオロジーの第８版に従シ）本図をサーモモノスポ
ラ・ビリディスと同定した。(3) Physiological properties ■ Growth temperature range 28-60℃ Optimum growth temperature range 45-50℃ ■ Liquefaction of gelatin
Positive ■ Hydrolysis of starch Positive (on starch yeast agar medium) ■ Coagulation of skim milk Positive〃 Peptonization Positive ■ Production of melanin pigment Negative Mycological (! On Determinative Bacteriology (Be
rgey's! , 4annual of Deter
Referring to the 8th edition of ``Minative Bacteriology'', the main garden was planted with Thermomonospora hyrizois.
It was identified as a type of ermomonospora viriclis. However, the Verge Eighth Manual on Systematics Bacteriology (Be
rgey's Manual of Systema
ticBacteriology) Volume 1 does not mention Thermomonospora viridis, but Thermomonospora viridis.
Viridis (Saccharomonospora v.
iridis), but since there is no detailed description of its mycological properties, it was published in accordance with the 8th edition of Burgeys's Manual on Deterministic Trees and Batteriology. The figure was identified as Thermomonospora viridis.
なお、上記菌は工業技術院微生物工業技術研究所に昭和
６３年ＩＯ月１４日に寄託され、その受託番号は微工研
菌寄１０３３９号（Ｆ Ｅ ＲＭ Ｐ−１０３３９）
である。The above bacterium was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on IO 14, 1985, and its accession number is FERRM P-10339.
It is.
本発明の新規な耐熱性α−アミラーゼＴＦ−３５の製造
法につきさらに詳しく説明するっ上記のようなサーモモ
ノスポラ、属に属するα−アミラーゼＴＦ−３５生産菌
を適当な培地に接種し、その生育温度の観点から２８〜
６０℃、好ましくは４０〜５０℃にて、２４〜４８時間
好気的に培養することにより培養液中に菌体外分泌型酵
素として該α−アミラーゼＴＦ−３５が生成蓄積される
。The method for producing the novel thermostable α-amylase TF-35 of the present invention will be explained in more detail. α-amylase TF-35 producing bacteria belonging to the genus Thermomonospora as described above are inoculated into an appropriate medium. From the point of view of growth temperature 28~
By culturing aerobically for 24 to 48 hours at 60°C, preferably 40 to 50°C, α-amylase TF-35 is produced and accumulated in the culture solution as an extracellular secreted enzyme.
本発明に用いられる培地には安価に入手し得る公知の各
種材料を使用することができる。例えば、窒素源として
は、コーン・ステイープ・リカーポリペプトン、大豆粕
、フスマ、肉エキス、酵母エキス、アミノ酸液などであ
り、炭素源としては、水飴、マルトース、各種デンプン
、可溶性デンプン、デンプン液化液、デキストリン、サ
イクロデキストリンなどである。Various known materials that can be obtained at low cost can be used for the culture medium used in the present invention. For example, nitrogen sources include corn staple liquor polypeptone, soybean meal, bran, meat extract, yeast extract, amino acid solution, etc., and carbon sources include starch syrup, maltose, various starches, soluble starch, starch liquefied liquid, Dextrin, cyclodextrin, etc.
また、これらの炭素源や窒素源の他に各種の塩、例えば
マグネシウム塩、カリウム塩、リン酸塩等のｇｆ４塩や
各種ビタミン類を必要により添加する。In addition to these carbon sources and nitrogen sources, various salts such as gf4 salts such as magnesium salts, potassium salts, and phosphates, and various vitamins are added as necessary.
本発明の上記方法において使用するのに適した培地は、
例えば、１％可溶性デンプン、０．５％ポリペプトン、
０．５％酵母エキス、０．１％に２１（ＰＯ，。Suitable media for use in the above method of the invention are:
For example, 1% soluble starch, 0.5% polypeptone,
0.5% yeast extract, 0.1% to 21 (PO,.
００２％Ｍｇ５Ｏ，・７Ｈ２０、を含有する液体培地で
ある。This is a liquid medium containing 002% Mg5O, 7H20.
本発明において有用な前記α−アミラーゼＴＦ−３５生
産菌は菌体外分泌型の酵素を産生ずる。The α-amylase TF-35 producing bacteria useful in the present invention produces an extracellular secretion type enzyme.
培養後培養上清中に蓄積される菌体外分泌型酵素は、粗
酵素液として遠心分離により図体を除去することにより
得られる。粗酵素液をそのまま用いることが経済的で有
利である。しかしこれをさらに精製して使用することも
できる。精製のために、例えば硫安等による塩析、エタ
ノール、アセトン、イソプロパツール等による溶媒沈澱
法、限外濾過法、イオン交換樹脂等による一般的な酵素
精製法を用′ｖ）ることができる。The extracellular secreted enzyme accumulated in the culture supernatant after culturing can be obtained as a crude enzyme solution by removing the cells by centrifugation. It is economical and advantageous to use the crude enzyme solution as it is. However, it can also be used after further purification. For purification, general enzyme purification methods such as salting out with ammonium sulfate, solvent precipitation with ethanol, acetone, isopropanol, etc., ultrafiltration, ion exchange resin, etc. can be used. .
以下に、本発明のα−アミラーゼの好ましい製造法の一
例を挙げて更に詳しく説明する。Below, one example of a preferred method for producing α-amylase of the present invention will be described in more detail.
好熱性放線菌サーモモノスポラ・ビリディスＴＦ−３５
株（微工研菌寄１０３３９号）を、１％可溶性デンプン
、１％ポリペプトン、０．１％ＫｘＨＰＯ＜、 ０．
０２％Ｉ（ｇ Ｓ ０４・７Ｈ２０を含む培地に植菌し
、４５℃にて４８時間好気的に培養して得ちれる培養液
を１０，０００Ｘｇ、４℃にて連続遠心して菌体を除き
、約Ｌ ＩＪットルの上澄液（粗酵素液）を得るっつい
で、該上澄液に固形硫酸アンモニウムを添加し、８０％
飽和とし、４℃で一夜放置するっ生じた沈殿を１０，０
００ｘｇ、４℃にて遠心して集め、５ ｍＭ ＣａＣｌ
２を含む２ＱｍＭ）リス・塩酸緩衝液（ｐＨ８，０）
に溶解させた後、回復（ｋ液に対して一夜４℃で透析す
る。透析により生じる沈澱は遠心分離により除き、得ら
れる上澄液を５ｒｎｋｉｃａｃβ２を含む１０ｍＭトリ
ス・塩酸援（封液（ｐＨ８，０）で平衡化したＤＥＡＥ
−セルロースカラムに吸着させ、０．０５ＭのＮａＣｉ
を含む上記と同様な緩衝液によって酵素を溶出させる。Thermophilic actinomycete Thermomonospora viridis TF-35
strain (Feikoken Hyokuyori No. 10339) was mixed with 1% soluble starch, 1% polypeptone, 0.1% KxHPO, 0.
The cells were inoculated into a medium containing 02% I (g S 04.7H20) and cultured aerobically at 45°C for 48 hours. After removing and obtaining a supernatant liquid (crude enzyme solution) of approximately L IJ liter, solid ammonium sulfate was added to the supernatant liquid, and 80%
The resulting precipitate was saturated and left overnight at 4°C.
Collected by centrifugation at 00xg, 4°C, and diluted with 5mM CaCl.
2QmM containing 2) Lis-HCl buffer (pH 8,0)
The precipitate produced by the dialysis was removed by centrifugation, and the resulting supernatant was diluted with 10 mM Tris/hydrochloric acid (sealed solution (pH 8, DEAE equilibrated with 0)
- 0.05M NaCi adsorbed onto a cellulose column
The enzyme is eluted with a buffer similar to that described above.
溶出した活性画分を集め平均分画分子ｉ５．０００の限
外濾過膜を用いてａ縮した後、０．２ Ｍ Ｎａｃβを
含む上記同様のトリス・塩酸緩衝液を用いて−ｐｉ透析
する。次いで、該透析酵素を同様にＱ、２ＭＮａＣｆを
含む上記トリス・塩酸緩衝液で平衡化したセファクリル
Ｓ−２００カラムを用いてゲル濾過に付し、活性画分を
集める。かくして得られるＲ製酵素はポリアクリルアミ
ドゲルディスク電気泳動的に単一であり、活性収率は約
１８％であった。The eluted active fractions are collected and a-condensed using an ultrafiltration membrane with an average fractional molecular weight i5.000, followed by -pi dialysis using the same Tris/HCl buffer as above containing 0.2 M Nacβ. Next, the dialyzed enzyme is subjected to gel filtration using a Sephacryl S-200 column equilibrated with the above Tris/HCl buffer containing Q and 2M NaCf, and the active fraction is collected. The R enzyme thus obtained was single in polyacrylamide gel disk electrophoresis, and the activity yield was about 18%.
このようにして得られる本発明の新規アミラーゼＴＦ−
３５は下記の理化学的性質を有する。The novel amylase TF- of the present invention thus obtained
35 has the following physical and chemical properties.
（イ）作用
アミロペクチン、アミロース、デンプン、グリコーゲン
及びそれらの部分加水分解物中のα−１，４−グルコピ
ラノシド結合からなる直鎖状構造を特異的に加水分解し
、主成分としてマルトースを生成する。(a) Action The linear structure consisting of α-1,4-glucopyranoside bonds in amylopectin, amylose, starch, glycogen and their partial hydrolysates is specifically hydrolyzed to produce maltose as the main component.
（ワ）基質特異性
マルトトリオース以上の重合度を有するα−１，４グル
コピラノシド結合からなる直鎮状マルトオリゴ糖及びサ
イクロデキストリンを加水分解し、プルラン及びパノー
スを加水分解しない。(iv) Substrate specificity Hydrolyzes straight malto-oligosaccharides and cyclodextrins consisting of α-1,4 glucopyranoside bonds having a degree of polymerization higher than that of maltotriose, but does not hydrolyze pullulan and panose.
（ハ） 至 適 ｐＨ５，５〜６，０
（功安定ｐＨ
４０℃、３時間の条件下でｐｆ１５．５〜９，０で安定
である。(c) Optimum pH 5.5 to 6.0 (Stable pH) Stable at pf 15.5 to 9.0 under conditions of 40°C and 3 hours.
（ネ）作用適温 約６０℃
（へ）失活温度
６０℃、１０分間の条件下ではｐＨ４及びｐＨ９で完全
に失活し、ｐＨ６，３０分間の条件下で７０℃で完全に
失活する。(f) Suitable temperature for action: about 60°C (v) Inactivation temperature: Completely inactivated at pH 4 and pH 9 under conditions of 60°C for 10 minutes, and completely deactivated at 70°C under conditions of pH 6 and 30 minutes.
（ト）温度安定性
ｐＨ６，３０分間の条件下では、５５℃まで安定である
。(g) Temperature stability: Stable up to 55°C under conditions of pH 6 and 30 minutes.
（チ）阻害、活性化及び安定化
本酵素は、銅、水銀、カドミウム、亜鉛、３価の鉄、Ｎ
−ブロモサクシニイミドで阻害され、カルシウムで安定
化されろう
（１月分子量〈ゲル濾過法）
１３．０００±２．０００
（ヌ）等電点（クロマトフオーカシング法）５．２
更に、α−アミラーゼＴＦ−３５の活性測定法並びに活
性表示法は以下の通りである。(H) Inhibition, activation and stabilization This enzyme contains copper, mercury, cadmium, zinc, trivalent iron, N
- It will be inhibited by bromosuccinimide and stabilized by calcium (1 month molecular weight (gel filtration method) 13.000 ± 2.000 (nu) Isoelectric point (chromato focusing method) 5.2 In addition, α - The method for measuring and displaying the activity of amylase TF-35 is as follows.
０．１Ｍの酢酸援衝液（ｐＨ６，０）に溶解させた１％
（’ｖＶ／Ｖ）のアミｏ−ス溶液０．３ ｍｌに酵素液
０．０５−を混合し、５５℃で１０分間反応させた後、
ＤＮＳ試薬〔ジェイ・アール・サムナー・ジー・イー・
ソマーズ、“ラボラトリ−イクスベリメンツ イン バ
イオロジカル ケミストリーアカデミツク プレス、ニ
ューヨーク （ホＲ。1% dissolved in 0.1M acetic acid buffer (pH 6,0)
('vV/V) of amio-se solution (0.3 ml) was mixed with 0.05 ml of enzyme solution, and after reacting at 55°C for 10 minutes,
DNS reagent [JR Sumner G.E.
Somers, “Laboratory Experiments in Biological Chemistry Academic Press, New York.
Ｓｕｍｎｅｒ、 Ｇ、 巳、 Ｓｏｍｅｒｓ、“Ｌａｂ
ｏｒａｔｏｒｙεｘｐｅｒ ｉｍｅｎｔｓ＋ｎ Ｂｉｏ
ｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ、 Ａｃａｄ
ｅｍｉｃ Ｐｒｅｓｓ。Sumner, G., Somers, “Lab
oratoryεxper iments+n Bio
Logical Chemistry, Acad
emic Press.
Ｎｅｗ Ｙｏｒｋ）、 ３４５５項、１９４４年］ ｌ
−を加えて反応を止める。次いで、沸騰水中で１０分間
加熱した後急冷し、５ｄの水を加えて充分に撹拌する。New York), Section 3455, 1944]
Add - to stop the reaction. Next, the mixture is heated in boiling water for 10 minutes and then rapidly cooled, and 5 d of water is added and thoroughly stirred.
同時に処理した０、３５ｍｆの０．５ｍｇ／ｍ！グルコ
ース溶液を標準とし、その着色度分光光度計を用いて５
００ｎｒｒｌで測定する５酵素活性の単位は前述の条件
下で１分間に１μｍｏｌｅのグルコースに相当する還元
糖を生成する酵素量を１単位とした。0.5mg/m of 0 and 35mf treated at the same time! Using a glucose solution as a standard, its color intensity was measured using a spectrophotometer.
The unit of 5 enzyme activity measured at 00 nrrl is the amount of enzyme that produces reducing sugar equivalent to 1 μmole of glucose per minute under the above conditions.
本発明の方法によって得られる耐熱性α−アミラーゼＴ
Ｆ−３５の理化学的、酵禦化学的諸件賃と従来公知の放
線菌由来のマルトース生成型α−アミラーゼとの比較を
第１表に示す。Thermostable α-amylase T obtained by the method of the present invention
Table 1 shows a comparison of the physical and chemical properties of F-35 and the conventional maltose-producing α-amylase derived from actinomycetes.
作用
本発明のα−アミラーゼは、アミロース、アミロペクチ
ン、デンプン、グリコーゲン中のα−１゜４−グルコピ
ラノシド結合を加水分解し、マルトースを主成分とする
糖質を生成する。このマルトースはデンプン糖の一種で
麦芽糖ともよばれ、般に甘味剤、栄養剤などとして有用
なものである。Effect: The α-amylase of the present invention hydrolyzes α-1°4-glucopyranoside bonds in amylose, amylopectin, starch, and glycogen to produce carbohydrates whose main component is maltose. Maltose is a type of starch sugar, also called maltose, and is generally useful as a sweetener, nutritional agent, etc.
このデンプン糖の生成反応は、低温かつ弱酸性条件下で
実施された場合、既に述べたような微生物汚染に起因す
る様々な悪影響を受けることが知られている。そこで、
このデンプンの酵素加水分解反応をできるだけ高温条件
下で行ない、また、中性〜弱アルカリ側のｐＨ条件下で
実施することが望ましいとされている。It is known that when this starch sugar production reaction is carried out at low temperatures and under slightly acidic conditions, it is affected by various adverse effects caused by microbial contamination as described above. Therefore,
It is considered desirable to carry out this enzymatic hydrolysis reaction of starch under conditions as high as possible, and under pH conditions ranging from neutral to weakly alkaline.
しかしながら、これまでに単離されたマルトースを主に
生成するα−アミラーゼは熱安定性に劣り、殆どが５０
℃前後の至適温度を示すにすぎなかった。更に、一部高
温領域に至適温度を有するものも知られているが、酵素
の生産性が悪い、該酵素産生微生物の培養が困難である
等の欠点を有しており、高価であり、大規模使用ができ
ないなど工業的観点から十分満足できるものでなかった
。However, the α-amylases that mainly produce maltose that have been isolated so far have poor thermostability, and most
It only showed an optimum temperature of around ℃. Furthermore, some products having an optimum temperature in the high temperature range are known, but they have drawbacks such as poor enzyme productivity and difficulty in culturing the enzyme-producing microorganisms, and are expensive. It was not fully satisfactory from an industrial point of view, as it could not be used on a large scale.
しかしながら、このようなデンプンの酵素分解反応に係
わる諸問題点は、本発明によるα−アミラーゼ並びにそ
の製法によってほぼ解決される。However, these problems related to the enzymatic decomposition reaction of starch can be almost solved by the α-amylase and the method for producing the same according to the present invention.
即ち、まず本発明のα−アミラーゼは約６０℃に酉￥素
反応の至適温度を有しており、至適ｐＨも弱酸性〜中性
領域にある。従って、デンプンの加水分解を高温でしか
も弱酸性領域近傍で実施することが可能となる。この事
実は、微生物汚染を確実に防止することを保証し、また
汚染に基づ＜ｐＨ低下を補償する目的でアルカリ試薬を
添加する必要もなくなり、コストパフォーマンスにおけ
る大巾な改善が期待できる。更に、高温での反応が可能
なことから、反応速度の改善が期待でき、これは分解生
成物の量産性を保証する。That is, firstly, the α-amylase of the present invention has an optimum temperature for the reaction at about 60° C., and an optimum pH in the weakly acidic to neutral range. Therefore, starch hydrolysis can be carried out at high temperatures and in the vicinity of the weakly acidic region. This fact ensures that microbial contamination is reliably prevented, and also eliminates the need to add alkaline reagents to compensate for pH decreases due to contamination, and a significant improvement in cost performance can be expected. Furthermore, since the reaction can be carried out at high temperatures, the reaction rate can be expected to be improved, which guarantees mass production of decomposition products.
かくして、本発明の方法によればデンプンの酵素生産の
ために有用α−アミラーゼを安価かつ大量に供給でき、
またかくして得られる酵素によりマルトースを高度に含
有するデンプン塘の工業的に大規模な生成を有利に実施
できる。Thus, according to the method of the present invention, α-amylase useful for enzyme production of starch can be supplied at low cost and in large quantities;
The enzyme thus obtained also allows advantageous industrial large-scale production of starch barrels containing a high degree of maltose.
実施例
以下、実施例に従って本発明の新規耐熱性マルトース生
成型のα−アミラーゼＴＦ−３５の製造法及びそれを用
いたデンプンまたはその分解生成物からのを用な糖類の
製造法につき更に具体的に説明する。EXAMPLES Hereinafter, in accordance with the Examples, the method for producing the novel heat-stable maltose-producing α-amylase TF-35 of the present invention and the method for producing saccharides from starch or its decomposition products using the same will be described in more detail. Explain.
しかしながら、本発明の範囲は以下の実施例によって回
答制限されるものはない。However, the scope of the present invention is not limited by the following examples.
実施例１
好熱性放線菌サーモモノスポラ・ビリディスＴ Ｆ −
３５（Ｔｈｅｒｍｏｍｏｎｏｓｐｏｒａ ｖｉｒｉｄｉ
ｓＴ Ｆ −３５）菌株を５００ｍ！容の三角フラスコ
中に可溶性デンプン２％、ゼラチン加水分解物２％、Ｋ
、ＨＰＯ，０，１％、Ｍｇ５ＣＬ ０．０２％を含む培
地（ｐＨ７，０） ５０ｍ１：の２０本に植菌し、４
５℃で４８時間２００ｐｐｍで回転振盪培養した後、集
菌しついで１２． ＯＯＯ×ｇ、４℃で３０分間遠心分
離して菌体を除き、１１０車位／ｒｎｌの酵素液８４０
ａｌｌ！得た。Example 1 Thermophilic actinomycete Thermomonospora viridis TF −
35 (Thermomonospora viridi
sT F-35) strain 500m! 2% soluble starch, 2% gelatin hydrolyzate, K
, HPO, 0.1%, and Mg5CL 0.02% (pH 7.0) were inoculated into 20 bottles of 50ml.
After culturing with rotary shaking at 200 ppm for 48 hours at 5°C, the bacteria were harvested and 12. Centrifuge at OOO×g for 30 minutes at 4°C to remove bacterial cells, and add 110 cells/rnl of enzyme solution 840
All! Obtained.
得られた粗酵素液に固形硫酸アンモニウムを添加し、８
０％飽和とし、４℃で一夜放置するユ生じた沈殿を１０
，０００ｘｇ、４℃にて遠心して集め、５ ｍＭ Ｃａ
Ｃｆｌ ２を含む２０ｍＭトリス・塩酸緩衝液（、ｐｌ
（８，０）に溶解させた後、同緩衝液に対して一夜４℃
で透析する。透析により生じる沈澱は遠心分離により除
き、得られる上澄液を５ｍＭＣａＣ１２を含む１０ｍＭ
）リス・塩酸緩衝液（ｐｌ（８，０）で平衡化したＤＥ
ＡＥ−セルロースカラムに吸着させ、０．０５ＭのＮａ
Ｃ１を含む上記と同様な緩衝液によって酵素を溶出させ
る。溶出した活性画分を集め平均分画分子ｆｆ１５．０
００の限外濾過膜を用いて濃縮した後、０．２ＭＮａＣ
ｊ７を含め上記同様のトリス・塩酸緩衝液を用いて一夜
透析する。Solid ammonium sulfate was added to the obtained crude enzyme solution, and 8
The resulting precipitate was adjusted to 0% saturation and left overnight at 4°C.
,000xg, 4°C, collected by centrifugation, 5mM Ca
20mM Tris-HCl buffer containing Cfl2 (, pl
(8,0) in the same buffer overnight at 4°C.
Dialyze with The precipitate produced by dialysis was removed by centrifugation, and the resulting supernatant was diluted with 10mM containing 5mM CaC12.
) DE equilibrated with Lis-HCl buffer (pl(8,0))
Adsorbed onto AE-cellulose column and 0.05M Na
The enzyme is eluted with a similar buffer as above containing C1. The eluted active fractions were collected and the average fractional molecule ff15.0
After concentration using a 0.00 ultrafiltration membrane, 0.2M NaC
Dialyze overnight using the same Tris/HCl buffer as above, including J7.
次いで、該透析酵素を同様に０．２ＭＮａＣβを含む上
記トリス・塩酸緩衝液で平衡化したセファクリルＳ−２
００カラムを用いてゲル濾過に付し、活性画分を集める
。かくして得られる精製α−アミラーゼＴＦ−３５はポ
リアクリルアミドゲルディスク電気泳動的に単一であり
、活性収率は約１８％であった。Next, the dialyzed enzyme was treated with Sephacryl S-2 which had been equilibrated with the above Tris/HCl buffer containing 0.2M NaCβ.
The active fractions are collected by gel filtration using a 00 column. The thus obtained purified α-amylase TF-35 was single in polyacrylamide gel disk electrophoresis, and the activity yield was about 18%.
実施例２
（イ）１０ｍ〜□ｒ ＣａＣ１’ｚ ・２Ｈ２０を含
む５ｍｆ！の５％（Ｗ／Ｖ）の可溶性デンプン（メルク
社製）にデンプンＩｇ当り２００単位の実施例１で得た
本発明の精製α−アミラーゼＴＦ−３５を添加し、 ５
５℃、ｐ）ｔ５．５で反応させた。２４時間後の反応液
中の糖組成をバイオラッド（Ｂｉｏ−Ｒａｄ−Ｌａｂ）
社製ＨＰＸ−４２Ａカラムを用いる高速液体クロマトグ
ラフ法（ＨＰＬＣ）で測定した。Example 2 (a) 5mf including 10m~□r CaC1'z・2H20! 200 units of purified α-amylase TF-35 of the present invention obtained in Example 1 per Ig of starch was added to 5% (W/V) of soluble starch (manufactured by Merck & Co., Ltd.).
The reaction was carried out at 5°C, p)t5.5. The sugar composition in the reaction solution after 24 hours was measured using Bio-Rad Lab.
It was measured by high performance liquid chromatography (HPLC) using a HPX-42A column manufactured by Co., Ltd.
その結果、グルコース１２．５％、フルドース７３．４
％、マルトトリオース８．２％、オリゴ糖５．９％であ
った。As a result, glucose 12.5%, full dose 73.4
%, maltotriose 8.2%, and oligosaccharide 5.9%.
（ロ）特異性
マルトース、マルトトリオース、マルトテトラオース、
マルトペンタオース、マルトヘキサオース、パノース、
プルラン、及びα、β、Ｔ−サイクロデキストリンの１
０ ｍＭ ＣａＣρ２・２Ｈ２０を含む５％溶液５ｍｌ
に基質１ｇにつき２００単位の本発明の精製α−アミラ
ーゼＴＦ−３５を添加し、５５℃で２４時間反応させた
。反応生成物をＩＩＰＬｃ法で測定した結果を第２表に
示す。(b) Specificity maltose, maltotriose, maltotetraose,
maltopentaose, maltohexaose, panose,
Pullulan and α, β, T-cyclodextrin 1
5ml of 5% solution containing 0mM CaCρ2.2H20
200 units of purified α-amylase TF-35 of the present invention was added per 1 g of substrate, and the mixture was reacted at 55° C. for 24 hours. Table 2 shows the results of measuring the reaction product using the IIPLc method.
（ハ）至適ｐＨ
Ｏ４ＩＭ酢酸護衝液（ｐＨ４，０，５，０，５，５゜６
．０）、０．ＩＭリン酸謹衝液（ｐＨ６．０ 、 ６．
５７．０，８．０＞または、０．１Ｍグリシン援衝液（
ｐＨ９，０）に溶解させた１ ％（Ｗ／Ｖ） 可ｉ？ｌ
Ｅヂンブンイ容液１．０ｄに本発明の精製酵素を予め１
ｍ〜Ｉ ＥＤＴＡを含む水で一夜透析したα−アミラー
ゼ含有溶液０．０５ｍを混合し先に述べた方法でα−ア
ミラーゼ活性を測定した。α−アミラーゼＴＦ−３５の
至適ｐ）ＩはｐＨ６，０における酵素活性を１００とす
る相対活性で示した（第１図）。(c) Optimum pH O4IM acetic acid protective buffer solution (pH 4, 0, 5, 0, 5, 5゜6
．． 0), 0. IM phosphoric acid buffer solution (pH 6.0, 6.
57.0, 8.0> or 0.1M glycine buffer solution (
1% (W/V) dissolved in pH 9,0) Possible? l
Add 1.0 d of the purified enzyme of the present invention to 1.0 d of Ejinbunji solution in advance.
m-I 0.05 m of an α-amylase-containing solution dialyzed overnight against water containing EDTA was mixed and α-amylase activity was measured by the method described above. The optimum p)I of α-amylase TF-35 is expressed as a relative activity with the enzyme activity at pH 6.0 as 100 (Figure 1).
（ニ）安定ｐＨ
ｐＨ４〜９の範囲で（ハ）で用いた酵素と同様に処理し
た本発明のα−アミラーゼＴＦ−３５を４５℃で３時間
及び６０℃で１０分間処理し、ｐＨ６，０で処理した時
の残存酵素活性を１００とする相対活性を第２図に示す
。なお、使用した暖衡液は酢酸（ｐＨ４〜６）、リン酸
（ｐＨ６〜８）及びグリシ：／−ＮａＯＨ−ＮａＣ；！
（ｐｉ（９）である。(d) Stable pH: The α-amylase TF-35 of the present invention, which had been treated in the same way as the enzyme used in (c), was treated at 45°C for 3 hours and at 60°C for 10 minutes, and the pH was adjusted to pH 6.0. The relative activity is shown in FIG. 2, taking the residual enzyme activity when treated with 100 as 100. The warm and balanced solutions used were acetic acid (pH 4-6), phosphoric acid (pH 6-8), and glycylate:/-NaOH-NaC;!
(pi(9).
ｐＨ６，３０分、７０℃、
〈ネ）作用適温
くハ）で用いた酵素と同様に処理した本発明のα−アミ
ラーゼＴＦ−３５含有溶液の活性を各温度で測定した。The activity of the α-amylase TF-35-containing solution of the present invention, which was treated in the same manner as the enzyme used in (c), at pH 6, 30 minutes, 70°C, (d) at an appropriate temperature, was measured at each temperature.
６０℃で測定した時の酵素活性を１００とした時の相対
活性を第３図に示す。Figure 3 shows the relative activity when the enzyme activity measured at 60°C is set as 100.
（へ）失活条件及び、温度安定性
精製した本発明のα−アミラーゼＴＦ−３５を（ハ）で
用いた酵素と同様に処理した後、１０ｍＭｃａｃ ｊｉ
！ ２ ・２Ｈ２０存在下及び、不存在下で５０℃及び
、６０℃で３０．６０，１４０，２１．０分間処理し、
残存する活性を測定した。その結果を第４図に示す。(f) Inactivation conditions and temperature stability After treating the purified α-amylase TF-35 of the present invention in the same manner as the enzyme used in (c), 10 mM cac ji
! 2. Treated at 50°C and 60°C for 30.60, 140, and 21.0 minutes in the presence and absence of 2H20,
The remaining activity was measured. The results are shown in FIG.
（チ）阻害化
（ハ）で用いた酵素と同様に処理した本発明のα−アミ
ラーゼＴＦ−３５含有溶液に總濃度１ｍＭ（水銀は０，
１ｍＭ）となるように各種金属の塩酸塩を添加し活性を
測定した。無添加時の活性を１００とした時の相対活性
を測定した結果を第３表に示す。(H) Inhibition (Hg: 0,
Hydrochloride salts of various metals were added to the solution to give a concentration of 1mM), and the activity was measured. Table 3 shows the results of measuring the relative activity when the activity without addition was set as 100.
第３表 金属及び化学試葵の影響
くり〉 分 子 １１
セファクリルＳ−２００を用いたゲル濾過法により求め
た本発明のα−アミラーゼＴＦ−３５の分子量は、１３
．０００±２．０００であった（第５図）Ｃ
（ヌ）等電点
ファルマシア社１１ＰＢＥ９４ゲル及び、ポリバッファ
ー７４を用いるクロマトフオーカシング法により求めた
本発明のα−アミラーゼＴＦ−３５の等電点は、５．２
であった（第６図）Ｃ実施例３
好熱性放線菌サーモモノスポラ・ビリディスＴ Ｆ −
３５＜Ｔｈｅｒｍｏｍｏｎｏｓｐｏｒａ ｖｉｒｉｄｉ
ｓ Ｔ Ｆ −３５）菌株を５００社容０三角フラスコ
中の可溶性デンプン２％、ゼラチン加水分解物２％、Ｋ
、ＨＰＯ４０，１％、：、４ｇ５Ｏ，０，０２％を含む
培地（ｐＨ７．０） ５０ｒｎｌの１０本に植菌し、
４５℃で４８時間、２００ｒ、 ｐｌｍ、で回転振盪培
養した後、集菌しついで１２．０００ｇ、４℃で３０分
間遠心分離して菌体を除き、８７．５単位／−の粗酵素
液４５０−を得た。ついで平均分画分子量５．０００の
限外濾過膜を用いてこれを’４１ Ｒし、３．２００単
位／蔵のａ１粗酵素液を約１０ｍ１得た。なお、酵素の
活性単位は上記方法によって測定した。Table 3 Influence of metal and chemical assays> Molecule 11 The molecular weight of α-amylase TF-35 of the present invention determined by gel filtration using Sephacryl S-200 is 13
．． 000±2.000 (Figure 5) The electric point is 5.2
(Figure 6)C Example 3 Thermophilic actinomycete Thermomonospora viridis TF-
35<Thermomonospora viridi
s T F-35) strain 2% soluble starch, 2% gelatin hydrolyzate, K
, HPO 40.1%, :, 4g 5O, 0.02% medium (pH 7.0) inoculated into 10 50rnl bottles,
After culturing with rotational shaking at 200r, plm for 48 hours at 45°C, the bacteria were collected and centrifuged at 12,000g for 30 minutes at 4°C to remove bacterial cells, and 87.5 units/- of crude enzyme solution was obtained. I got -. This was then subjected to '41 R using an ultrafiltration membrane with an average molecular weight cut off of 5.000 to obtain about 10 ml of a1 crude enzyme solution containing 3.200 units/cell. In addition, the activity unit of the enzyme was measured by the above method.
ついで、５ｍｆの２０％（Ｗ／Ｖ）とうもろこしデンプ
ン懸濁液を細菌液化型耐熱性α−アミラーゼ（大和化成
側型、タライスターゼＴ−５）を用いて１０５℃で液化
した後、直に１２５℃で３０分間オートクレーブし、Ｄ
、 Ｅ （ＤｅｘｔｒｏｓｅＥｑｕｉｖａｌｅｎｔ
：直接還元糖に対する全固定物の割合）８のデンプン液
化液を得た。該デンプン液化液に対しデンプン１ｇ当り
２００単位のＴＦ’−３５菌の前述の方法で調製した濃
縮粗酵素液を添加し、ｐＨ５，５，５５℃で４８時間反
応させた。Next, 5mf of 20% (W/V) corn starch suspension was liquefied at 105°C using a bacterial liquefaction type heat-stable α-amylase (Yamato Kasei side type, Talaistase T-5), and then directly heated to 125°C. Autoclave for 30 minutes at
, E (DextroseEquivalent
: Ratio of total fixed substances to direct reducing sugars) A starch liquefied solution of 8 was obtained. To the starch liquefaction solution, 200 units/g of starch of concentrated crude enzyme solution of TF'-35 bacteria prepared by the above method was added, and the mixture was reacted at pH 5, 5, and 55° C. for 48 hours.
ついで、反応柊了後の糖液に対して、０．２％（Ｗ／Ｖ
）の活性炭を添加し、沸騰水浴中で５分間加熱した後、
０．４５μｍポアサイズのメンブランフィルタ−で濾別
して得られる糖液中のマルトース含量をアミネックス（
Ａｍ１ｎｅｘ ）ＨＰ Ｘ −４２Ａ （Ｂｉｏ −Ｒ
ａｄ −Ｌａｂ社製）カラムを用いる高速液体クロマド
グガフ法で測定した結果、７２．５％であった。Next, 0.2% (W/V) was added to the sugar solution after the reaction.
) and after heating in a boiling water bath for 5 minutes,
Aminex (
Am1nex) HP X-42A (Bio-R
As a result of measurement using a high performance liquid chromatographic method using a column (manufactured by ad-Lab), it was 72.5%.
実施例４
５ｍ１の２０％（Ｗ／Ｖ）馬鈴薯デンプン懸濁液を細菌
液化型α−アミラーゼ（大和化成■製りライスターゼし
−１）を用いて９０℃で液化した後、直ちに１２５℃で
３０分間オートクレーブし、Ｄ。Example 4 5ml of 20% (W/V) potato starch suspension was liquefied at 90°C using bacterial liquefied α-amylase (Lystase Shi-1 manufactured by Daiwa Kasei), and then immediately heated to 125°C for 30 minutes. Autoclave for minutes, D.
Ｅ９のデンプン液化液を得た。A starch liquefied liquid of E9 was obtained.
該デンプン液化液の固形物に対し、０．２％の大豆由来
のβ−アミラーゼ（長潮生化学社製１０．０００Ｕ／ｇ
） 及び実施例３で調整した濃縮粗酵素液をデンプン
Ｉｇ当り２００単位添加し、ｐＨ５，５，５５℃で４８
時間反応させた。0.2% of soybean-derived β-amylase (manufactured by Nagashio Seikagaku Co., Ltd., 10.000 U/g) was added to the solid matter of the starch liquefied liquid.
) and the concentrated crude enzyme solution prepared in Example 3 were added at 200 units per Ig of starch, and the mixture was heated at pH 5, 5, and 48°C at 55°C.
Allowed time to react.
ついで得られる糖液を実施例３と同様の方法で脱色、濾
過し、糖液中のマルトースの含有量を高速液体クロマト
グラフ法で測定した結果、８２．３％であった。The resulting sugar solution was then decolorized and filtered in the same manner as in Example 3, and the maltose content in the sugar solution was measured by high performance liquid chromatography, and was found to be 82.3%.
実施例５
１０ｍ１の２５％（Ｗ／Ｖ）とうもろこしデンプン懸濁
液を細菌液化型α−アミラーゼ（大和化成Ｇａ製クりイ
スターゼし−５）を用し）で９０℃で液化した後、直ち
に１２５℃でオートクレーブし、Ｄ、Ｅ５のデンプン液
化液を得た。Example 5 10ml of 25% (W/V) corn starch suspension was liquefied at 90°C using bacterial liquefaction α-amylase (Krystase Shi-5 manufactured by Daiwa Kasei Ga), and then immediately It was autoclaved at ℃ to obtain starch liquefied liquids D and E5.
該デンプン液化液の固形物に対し、０．０５％プルラナ
ーゼ（大野製菓製３．８３０ Ｕ／ｇ）及び実施例３で
調整した濃縮粗酵素液をデンプン１ｇ当り３００単位添
加し、ｐＨ５．５．５５℃で４８時間反応させた。0.05% pullulanase (manufactured by Ohno Seika Co., Ltd., 3.830 U/g) and 300 units of the concentrated crude enzyme solution prepared in Example 3 were added to the solid matter of the starch liquefaction solution, and the pH was adjusted to 5.5. The reaction was carried out at 55°C for 48 hours.
つ５１で得られる糖液を実施例３と同様の方法で脱色、
濾過し、糖液中のマルトースの含有量を高速液体クロマ
トグラフ法で測定した結果、７８．２％であった。The sugar solution obtained in Step 51 was decolorized in the same manner as in Example 3.
After filtration, the maltose content in the sugar solution was measured by high performance liquid chromatography, and the result was 78.2%.
実施例６
実施例５で調製したデンプン液化液に固形物に対し、０
．０１％イソアミラーゼ（材厚製１２５０．０００ｔＪ
／ｇ＞及び実施例３で調製した濃縮粗酵素液をデンプン
１ｇ当り２００亘位添加し、ｐＨ５，５，５５℃で反応
させた。Example 6 The starch liquefied liquid prepared in Example 5 contained 0
．． 01% isoamylase (1250.000tJ made of material
/g> and the concentrated crude enzyme solution prepared in Example 3 was added in an amount of about 200 per gram of starch, and reacted at pH 5, 5, and 55°C.
ついで得られる糖液を実施例３と同様の方法で脱色、濾
過し、糖液中のマルトースの含有量を高速液体クロマト
グラフ法で測定した結果、７６．５％であった。The resulting sugar solution was then decolorized and filtered in the same manner as in Example 3, and the maltose content in the sugar solution was measured by high performance liquid chromatography, and was found to be 76.5%.
実施例７
約４５βの２８％（Ｗ／Ｖ）とうもろこしデンプン懸濁
液を細菌糖化型α−アミラーゼ（大和化成潤製クライス
ターゼし−５）を用いて９０℃で液化した後、直ちに１
２５℃でオートクレーブし、Ｄ、Ｅ５のデンプン液化液
を得た。Example 7 A 28% (W/V) corn starch suspension of approximately 45β was liquefied at 90°C using bacterial saccharification α-amylase (Clystase Shi-5 manufactured by Daiwa Kasei Jun), and then immediately
It was autoclaved at 25°C to obtain starch liquefied liquids D and E5.
該デンプン液化液の固形物に対し、０．１大豆由来のβ
−アミラーゼ（長潮生化学社製Ｌ０，０００Ｕ／ｇ）、
０．１％のプルラナーゼ（大野製薬製３、８３０ Ｕ／
ｇ）及び実施例３で調製したａ１粗酵素液をデンプン１
ｇ当り１００単位添加し、ｐＨ５，５，５５℃で４８時
間反応させた。0.1 β derived from soybean to the solid matter of the starch liquefied liquid
- amylase (manufactured by Nagashio Biochemical Co., Ltd. L0,000U/g),
0.1% pullulanase (Ono Pharmaceutical 3, 830 U/
g) and the a1 crude enzyme solution prepared in Example 3.
100 units per g were added, and the reaction was carried out at pH 5, 5, and 55° C. for 48 hours.
ついで得られる糖液を実施例３と同様の方法で脱色、濾
過し、糖液中のマルトースの含有量を高速液体クロマト
グラフ法で測定した結果、８８．０％であった。The resulting sugar solution was then decolorized and filtered in the same manner as in Example 3, and the maltose content in the sugar solution was measured by high performance liquid chromatography, and was found to be 88.0%.
以上詳しく述べたように、本発明によれば高温度域（６
０℃）に酵素反応の至適温度を有し、しかも弱酸性〜弱
塩基性領域に安定ｐＨ範囲を有するマ）レトース生成型
α−アミラーゼが提供され、このものはその前記緒特性
に基づき、デンプンの酵素分解反応の収率、効率を大幅
に改善することを可能とする。しかも余分な試薬の必要
性を排除し、脱塩などの後処理工程が短縮でき経済性も
著しく向上する。As described above in detail, according to the present invention, the high temperature range (6
Provided is a ma)letose-producing α-amylase having an optimal temperature for enzymatic reaction at 0°C) and a stable pH range in the weakly acidic to weakly basic region, which is based on the above-mentioned characteristics, It makes it possible to significantly improve the yield and efficiency of starch enzymatic decomposition reactions. Moreover, it eliminates the need for extra reagents, shortens post-processing steps such as desalting, and significantly improves economic efficiency.
かくして、本発明は安価かつ大規模でのデンプン糖の製
造を可能とするもので工業的に極めて大きな意義を有す
るものである。Thus, the present invention enables the production of starch sugar at low cost and on a large scale, and has extremely great industrial significance.
第１図及び第３図には、それぞれ本発明のα−アミラー
ゼＴＦ−３５の相対活性のｐＨ依存性及び温度依存性を
示す。第２図及び第４図には、それぞれ本発明のα−ア
ミラーゼＴＦ−３５の残存活性とｐＨ及び温度との関係
を示す。第５図は、本発明のα−アミラーゼＴＦ−３５
を含むタンパク賃の分子量とフラクションとの関係を示
す図である。
第６図は、本発明のα−アミラーゼＴＦ−３５０等電点
が５．２であることを示す図である。FIGS. 1 and 3 show the pH dependence and temperature dependence of the relative activity of α-amylase TF-35 of the present invention, respectively. FIGS. 2 and 4 show the relationship between the residual activity of α-amylase TF-35 of the present invention and pH and temperature, respectively. FIG. 5 shows α-amylase TF-35 of the present invention.
FIG. 2 is a diagram showing the relationship between the molecular weight and fraction of a protein containing . FIG. 6 is a diagram showing that the isoelectric point of α-amylase TF-350 of the present invention is 5.2.
Claims (5)
−３５。 （イ）作用 アミロペクチン、アミロース、デンプン、 グリコーゲン及びそれらの部分加水分解物中のα−１，
４−グルコピラノシド結合からなる直鎖状構造を特異的
に加水分解し、主成分としてマルトースを生成する。 （ロ）基質特異性 マルトトリオース以上の重合度を有するα −１，４グルコピラノシド結合からなる直鎖状マルトオ
リゴ糖及びサイクロデキストリンを加水分解し、プルラ
ン及びパノースを加水分解しない。 （ハ）至適ｐＨ５．５〜６．０ （ニ）安定ｐＨ ４０℃、３時間の条件下でｐＨ５．５〜９．０で安定で
ある。 （ホ）作用適温約６０℃ （ヘ）失活温度 ６０℃、１０分間の条件下でｐＨ４及びｐＨ９で完全に
失活し、ｐＨ６、３０分間の条件下で７０℃で完全に失
活する。 （ト）温度安定性 ｐＨ６、３０分間の条件下では、５５℃まで安定である
。 （チ）阻害、活性化及び安定化 本酵素は、銅、水銀、カドミウム、亜鉛、 ３価の鉄、Ｎ−ブロモサクシニイミドで阻害され、カル
シウムで安定化される。 （リ）分子量（ゲル濾過法） １３，０００±２，０００ （ヌ）等電点（クロマトフォーカシング法）５．２(1) α-amylase TF with the following physical and chemical properties
-35. (b) Effect α-1 in amylopectin, amylose, starch, glycogen and their partial hydrolysates,
It specifically hydrolyzes the linear structure consisting of 4-glucopyranoside bonds to produce maltose as the main component. (b) Substrate specificity Hydrolyzes linear malto-oligosaccharides and cyclodextrins consisting of α-1,4 glucopyranoside bonds having a degree of polymerization higher than that of maltotriose, but does not hydrolyze pullulan and panose. (c) Optimum pH 5.5-6.0 (d) Stable pH Stable at pH 5.5-9.0 under conditions of 40°C and 3 hours. (e) Suitable action temperature: about 60°C (f) Inactivation temperature: Completely inactivated at pH 4 and pH 9 under conditions of 60°C for 10 minutes, and completely deactivated at pH 6 and 70°C for 30 minutes. (g) Temperature stability Stable up to 55°C under conditions of pH 6 and 30 minutes. (h) Inhibition, activation, and stabilization This enzyme is inhibited by copper, mercury, cadmium, zinc, trivalent iron, and N-bromosuccinimide, and stabilized by calcium. (li) Molecular weight (gel filtration method) 13,000±2,000 (n) Isoelectric point (chromatofocusing method) 5.2
アミラーゼＴＦ−３５。(2) The α- according to claim (1), which is an exocrine enzyme.
Amylase TF-35.
ｒａ）属に属するα−アミラーゼＴＦ−３５生産菌を培
養し、培養物からα−アミラーゼＴＦ−３５を採取する
ことを特徴とするα−アミラーゼＴＦ−３５の製造法。(3) Thermomonospora
A method for producing α-amylase TF-35, which comprises culturing α-amylase TF-35-producing bacteria belonging to the genus ra) and collecting α-amylase TF-35 from the culture.
ｒａ）属に属するα−アミラーゼＴＦ−３５生産菌が微
工研菌寄第１０３３９号である請求項（３）記載の製造
法。(4) Thermomonospora
The production method according to claim (3), wherein the α-amylase TF-35 producing bacterium belonging to the genus ra) is Kaikoken Bibori No. 10339.
単独もしくはβ−アミラーゼ及び／又は枝切り酵素と併
用して、デンプンもしくはその加水分解物に作用させる
ことを特徴とする糖類の製造法。(5) Production of a saccharide, characterized in that α-amylase TF-35 according to claim (1) is used alone or in combination with β-amylase and/or debranching enzyme to act on starch or its hydrolyzate. Law.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63265614A JP2691354B2 (en) | 1988-10-21 | 1988-10-21 | Novel α-amylase, method for producing the same and method for producing saccharide using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63265614A JP2691354B2 (en) | 1988-10-21 | 1988-10-21 | Novel α-amylase, method for producing the same and method for producing saccharide using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02113886A true JPH02113886A (en) | 1990-04-26 |
| JP2691354B2 JP2691354B2 (en) | 1997-12-17 |
Family
ID=17419578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63265614A Expired - Lifetime JP2691354B2 (en) | 1988-10-21 | 1988-10-21 | Novel α-amylase, method for producing the same and method for producing saccharide using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2691354B2 (en) |
-
1988
- 1988-10-21 JP JP63265614A patent/JP2691354B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP2691354B2 (en) | 1997-12-17 |
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