JPH0325155B2 - - Google Patents
Info
- Publication number
- JPH0325155B2 JPH0325155B2 JP4215988A JP4215988A JPH0325155B2 JP H0325155 B2 JPH0325155 B2 JP H0325155B2 JP 4215988 A JP4215988 A JP 4215988A JP 4215988 A JP4215988 A JP 4215988A JP H0325155 B2 JPH0325155 B2 JP H0325155B2
- Authority
- JP
- Japan
- Prior art keywords
- raw starch
- degrading enzyme
- starch
- enzyme
- 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
Links
- 108090000790 Enzymes Proteins 0.000 claims description 46
- 102000004190 Enzymes Human genes 0.000 claims description 46
- 229920002472 Starch Polymers 0.000 claims description 29
- 235000019698 starch Nutrition 0.000 claims description 29
- 239000008107 starch Substances 0.000 claims description 27
- 230000000593 degrading effect Effects 0.000 claims description 13
- 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 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 5
- 230000002779 inactivation Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 229920000858 Cyclodextrin Polymers 0.000 claims description 3
- 239000001116 FEMA 4028 Substances 0.000 claims description 3
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 claims description 3
- 229940043377 alpha-cyclodextrin Drugs 0.000 claims description 3
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 3
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 3
- 229960004853 betadex Drugs 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 229920001542 oligosaccharide Polymers 0.000 claims description 3
- 150000002482 oligosaccharides Chemical class 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 229940088598 enzyme Drugs 0.000 description 40
- 238000004440 column chromatography Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 241000193380 Bacillus sp. B1018 Species 0.000 description 4
- 240000007594 Oryza sativa Species 0.000 description 4
- 235000007164 Oryza sativa Nutrition 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000009566 rice Nutrition 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 229940099112 cornstarch Drugs 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000010025 steaming Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- XJEVHMGJSYVQBQ-VIFPVBQESA-N (1s)-2,3-dihydro-1h-inden-1-amine Chemical compound C1=CC=C2[C@@H](N)CCC2=C1 XJEVHMGJSYVQBQ-VIFPVBQESA-N 0.000 description 1
- 229940122816 Amylase inhibitor Drugs 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241001530056 Athelia rolfsii Species 0.000 description 1
- 241000193752 Bacillus circulans Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 101001120710 Homo sapiens Ovarian cancer G-protein coupled receptor 1 Proteins 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 102100026070 Ovarian cancer G-protein coupled receptor 1 Human genes 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000952054 Rhizopus sp. Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 239000003392 amylase inhibitor Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
（産業上の利用分野）
本発明は新規生澱粉分解酵素B1018、その製造
法及び生産菌に関するものである。
本発明の生澱粉分解酵素B1018は生澱粉をよく
分解するので、発酵原料や澱粉の分解に生澱粉分
解酵素B1018を使用すれば発酵原料や澱粉のα化
のための蒸煮工程を省略することができ、貴重な
エネルギーの節減につながり、発酵業界、澱粉業
界、糖業界等に益するところ大なるものがある。
（従来技術）
一般に、生澱粉は大きな結晶構造をもち、この
結晶構造のために澱粉分解酵素では容易に分解さ
れないと考えられてきた。
従来の発酵原料、糖原料としての生澱粉は蒸煮
してα化し、これに澱粉分解酵素を作用させて液
化、糖化し、発酵、糖製造に使用されている。
しかし、近年、蒸煮に要する燃料費の高騰から
生澱粉をそのまま酵素で分解し、発酵、糖製造等
に使用しようとする研究が進められて来た。
従来、生澱粉分解酵素としてはRhizopus sp.
の生産するグルコアミラーゼ、Penicillium
lanosum OGR−１の生産する生澱粉分解酵素、
Aspergillus Ｋ−27の生産するアミラーゼ、
Corticium rolfsiiの生産する生澱粉糖化酵素、
Bacillus circulans Ｆ−２の生産するアミラーゼ
などが広く知られるようになつた。
（発明が解決しようとする問題点）
従来知られている生澱粉分解酵素はいずれも生
澱粉の分解速度が遅かつたり、また、その大部分
がエキソ型酵素であるなど工業生産に使用するに
は問題が多かつた。
いまや、エネルギー節減のために、すぐれた生
澱粉分解酵素が待望されているのである。
（問題点を解決するための手段）
本発明者らは、工業的に使用して有用な生澱粉
分解酵素を得るために鋭意研究したところ、バチ
ルス属の新規分離株が著じるしくすぐれた生澱粉
分解酵素を生産することを知つた。
新たに単離した生澱粉分解酵素を酵素学的に調
べたところ、公知の生澱粉分解酵素とは相違する
新規な生澱粉分解酵素と認められ、この酵素を生
澱粉分解酵素B1018と命名するに至つた。
また、生澱粉分解酵素B1018を生産する菌は胞
子を形成し、周鞭毛を有し、運動性があるなどバ
チルス属に属するものとは認められたが、明確な
種を確定することはできず、本菌をバチルスsp.
B1018と命名した。そして、バチルスsp.B1018は
微工研菌寄第9840号として寄託された。
本発明は生澱物を分解し、オリゴ糖、マルトー
ス及びグルコースを生成し、かつ、α−サイクロ
デキストリン及びβ−サイクロデキストリンを分
解する生澱粉分解酵素B1018に関する。
また、本発明はバチルス属に属する生澱粉分解
酵素B1018生産菌を培養し、生澱粉分解酵素
B1018を採取することを特徴とする生澱粉分解酵
素B1018の製造法である。
更に、本発明は生澱粉分解酵素B1018を生産す
るバチルスsp.B1018に関するものである。
本発明の生澱粉分解酵素B1018の酵素学的性質
は次の通りである。
(1) 作用及び基質特異性
（作用）
生澱粉を分解し、オリゴ糖、マルトース、グ
ルコースを同時に生成する。また、α−サイク
ロデキストリン及びβ−サイクロデキストリン
を分解する。
（基質特異性）
一定量の酵素による各種生澱粉の分解度は次
の通りである。
即ち、基質を0.02Mリン酸緩衝液に懸濁し、
酵素を加え45℃、４時間反応後還元糖の生成量
を測定し、コーンを100とした相対活性で示す。
コーン 100
ワクシーコーン 103
タピオカ 89
ポテト 15
ライス 113
小 麦 97
(2) 至適PH及び安定PH範囲
至適PH：6.0±0.5
安定PH：4.5〜12
(3) 至適温度及び作用適温の範囲
至適温度：55℃
作用適温の範囲：40〜70℃
(4) PH、温度による失活条件
PH３以下、12以上で失活する
10ｍＭ CaCl2存在下で70℃、30分で失活
(5) 阻害、活性化及び安定化
Ｓ−AI〔アミラーゼ阻害剤、Agric.Biol.
Chem.、41、919（1977）〕により非きつ抗阻害
を受ける
Ca++による活性化及び安定化
(6) 分子量
78000（SDS−PAGE法による）
本発明の生澱粉分解酵素B1018はバチルスsp.
B1018によつて生産される。
バチルスsp.B1018の菌学的性質は次に示され
る。
(Field of Industrial Application) The present invention relates to a novel raw starch-degrading enzyme B1018, a method for producing the same, and a producing bacterium. The raw starch-degrading enzyme B1018 of the present invention decomposes raw starch well, so if raw starch-degrading enzyme B1018 is used to degrade fermentation raw materials and starch, the steaming step for gelatinization of fermentation raw materials and starch can be omitted. This will lead to the saving of valuable energy and will greatly benefit the fermentation industry, starch industry, sugar industry, etc. (Prior Art) Generally, raw starch has a large crystalline structure, and it has been thought that this crystalline structure is not easily decomposed by starch-degrading enzymes. Raw starch, which is conventionally used as a raw material for fermentation and sugar, is steamed to gelatinize, and then treated with starch-degrading enzymes to liquefy and saccharify, and then used for fermentation and sugar production. However, in recent years, due to the rising cost of fuel required for steaming, research has been carried out to decompose raw starch as it is with enzymes and use it for fermentation, sugar production, etc. Conventionally, the raw starch degrading enzyme was Rhizopus sp.
Glucoamylase produced by Penicillium
Raw starch-degrading enzyme produced by lanosum OGR-1,
amylase produced by Aspergillus K-27,
Raw starch saccharifying enzyme produced by Corticium rolfsii,
Amylase produced by Bacillus circulans F-2 has become widely known. (Problems to be solved by the invention) Conventionally known raw starch-degrading enzymes all have a slow rate of degrading raw starch, and most of them are exo-type enzymes, making them difficult to use in industrial production. had many problems. Nowadays, there is a long-awaited need for excellent raw starch-degrading enzymes to save energy. (Means for Solving the Problems) The present inventors conducted intensive research to obtain a raw starch-degrading enzyme that is useful for industrial use, and found that a new strain isolated from the genus Bacillus was significantly superior. I learned that it produces raw starch degrading enzyme. When the newly isolated raw starch-degrading enzyme was enzymatically examined, it was recognized as a new raw starch-degrading enzyme different from known raw starch-degrading enzymes, and this enzyme was named raw starch-degrading enzyme B1018. I've reached it. In addition, the bacteria that produces raw starch degrading enzyme B1018 was recognized to belong to the genus Bacillus, as it forms spores, has periflagella, and is motile, but the species could not be clearly determined. , this bacterium is Bacillus sp.
It was named B1018. Bacillus sp.B1018 was deposited as FIKEN Bacteria No. 9840. The present invention relates to raw starch-degrading enzyme B1018, which decomposes raw starch to produce oligosaccharides, maltose and glucose, and also decomposes α-cyclodextrin and β-cyclodextrin. In addition, the present invention involves culturing raw starch-degrading enzyme B1018-producing bacteria belonging to the genus Bacillus, and producing raw starch-degrading enzyme B1018.
This is a method for producing raw starch degrading enzyme B1018, which is characterized by collecting B1018. Furthermore, the present invention relates to Bacillus sp.B1018 that produces raw starch degrading enzyme B1018. The enzymatic properties of raw starch degrading enzyme B1018 of the present invention are as follows. (1) Action and substrate specificity (action) Decomposes raw starch and simultaneously produces oligosaccharides, maltose, and glucose. It also decomposes α-cyclodextrin and β-cyclodextrin. (Substrate specificity) The degree of decomposition of various raw starches by a fixed amount of enzyme is as follows. That is, the substrate was suspended in 0.02M phosphate buffer,
After adding the enzyme and reacting at 45°C for 4 hours, the amount of reducing sugar produced was measured and expressed as relative activity with Cohn as 100. Corn 100 Waxy corn 103 Tapioca 89 Potato 15 Rice 113 Wheat 97 (2) Optimal PH and stable PH range Optimum PH: 6.0 ± 0.5 Stable PH: 4.5 to 12 (3) Optimal temperature and action temperature range Optimum temperature : 55℃ Suitable temperature range for action: 40 to 70℃ (4) Inactivation conditions depending on PH and temperature Inactivation occurs at pH 3 or below and PH 12 or above Inactivation occurs at 70℃ for 30 minutes in the presence of 10mM CaCl 2 (5) Inhibition, Activation and stabilization S-AI [amylase inhibitor, Agric.Biol.
Chem., 41 , 919 (1977)] Activation and stabilization by Ca ++ (6) Molecular weight: 78,000 (by SDS-PAGE method) The raw starch-degrading enzyme B1018 of the present invention is a Bacillus sp.
Produced by B1018. The mycological properties of Bacillus sp.B1018 are shown below.
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】
生澱粉分解酵素B1018の製造に際しては、バチ
ルスsp.B1018を生育する培地に接種し、20〜50
℃で25〜40時間通気撹拌培養することによつて、
培地中に著量の生澱粉分解酵素B1018を生産させ
ることができる。
培地としては、グルコース、マルトース、澱
粉、コーンスターチ等の炭素源、ポリペプトン、
肉エキス、尿素、アンモニア、無機窒素化合物等
の窒素源、酵母エキス、コーンスチープリカーな
どの栄養素等から選ばれたものを適宜含むものが
よい。好ましくは、各種生澱粉のうちの１種また
は、数種を培地に添加するのがよい。
得られた培養液は菌体を分離し、上清に生澱粉
分解酵素B1018を含有しているので、上清をその
まま酵素液として使用することもできる。
また、酵素は上清から硫安沈澱法によつて沈澱
させて、透析したりして粗酵素を得ることがで
き、更には、透析内液をDEAE−セルロースカラ
ムクロマトグラフイー、セフアデツクスG75カラ
ムクロマトグラフイー、CMセルロースカラムク
ロマトグラフイーなどにかけて精製酵素を得るこ
ともできる。
次に本発明の実施例、使用例を示すが、α−ア
ミラーゼ活性は不破の方法〔（Fuwa、J.
Biochem.41、583 1954）〕に従つて比活性として
測定した。タンパク質の測定はLowryの方法
〔O.H.Lowry etal.、J.Biol.Chem.193、265
（1951）〕により、牛血清アルブミンを標準として
測定した。
実施例 １
バチルスsp.B1018、FERM Ｐ−9840を500ml
三角フラスコ10本中で、グルコース1.0％、ポリ
ペプトン1.0％、生コーンスターチ0.5％、肉エキ
ス0.5％、酵母エキス0.2％（PH7.0）の組成を有す
る培地100mlにそれぞれ植菌し、45℃、36時間振
盪培養した。
得られた培養液を遠心分離（10000rpm、15分）
して菌体を除き培養上清820mlを得た。
この培養上清に496ｇの硫酸アンモニウムを加
え、0.8飽和とし、４℃で一液放置後、タンパク
質を沈殿させ1.13ｇ（比活性580U／mg protein）
を得た。
得られたタンパク質を５ｍＭトリス・塩酸緩衝
液（PH8.0）に溶解し、50ｍＭトリス・塩酸緩衝
液（PH8.0）＋２ｍＭ塩化カルシウムに対し透析し
た。
この透析内液をDEAE−セルロースカラムクロ
マトグラフイーにかけ吸着させ、50ｍＭトリス・
塩酸（PH8.0）で溶出する画分を回収した。この
回収画分92mlに硫酸アンモニウム56ｇ（0.8飽和）
を加え、４℃、12時間放置後、タンパク質を沈殿
させた。
得られたタンパク質を更に50ｍＭトリス・塩酸
緩衝液（PH5.6）＋２ｍＭ塩化カルシウム溶液に対
し透析した。
透析内液を更にセフアデツクスG75カラムクロ
マトグラフイーにかける。次いで活性画分に0.8
飽和になるように硫安を加えタンパク質を沈殿さ
せた。
得られたタンパク質を10ｍＭ・塩酸緩衝液（PH
5.6）に溶解し、同一緩衝液に対し透析し透析内
液をCM−セルロースカラムクロマトグラフイー
にかけ吸着させ食塩の濃度勾配（０−0.3M）を
有する10ｍＭ酢酸緩衝液で溶出し、活性画分を集
め、精製生澱粉分解酵素B1018 6.6mg（収率15.6
％、比活性15400U／mg protein）を得た。
実施例 ２
実施例１で得られた硫酸アンモニウム0.8飽和
沈澱物を透析後、DEAE−セルロースカラムクロ
マトグラフイーにかけ、第１図の溶出曲線を得
た。
第１図におけるフラクシヨンNo.３〜15を集め、
更に硫酸アンモニウム0.8飽和でタンパク質を沈
殿させ、透析後、セフアデツクスG75カラムクロ
マトグラフイーにかけ、第２図の溶出曲線を得
た。第２図におけるフラクシヨンNo.25〜45を集
め、再度硫酸アンモニウム0.8飽和で沈殿させ、
透析後CMセルロースカラムクロマトグラフイー
にかけ、第３図の溶出曲線を得た。第３図におけ
るフラクシヨンNo.35〜39を集め、凍結乾燥し、精
製生澱粉分解酵素B1018粉末を得た。
使用例 １
精白生米の粉末１ｇ（澱粉77.4％、水分14.5
％、その他8.1％）をリン酸緩衝液500mlに懸濁
し、実施例１で得られた生澱粉分解酵素B1018を
10mg加え撹拌しながら、45℃、４時間反応させ
た。対照として、同様に生米粉末１ｇを0.02Mリ
ン酸緩衝液500mlに懸濁し撹拌しながら45℃、４
時間反応させた。
反応終了後、試験、対照各々の区分の反応液を
10000rpm、５分遠心分離することによつて上澄
液480mlを沈澱物に分けた。
各々の上澄はベーリンガー・マンハイム山之内
(株)製のグルコース測定キツト（製品番号716251）
でグルコース量を測定した。沈澱物は100℃、２
時間乾燥し、未分解部分の乾燥重量を測定した。
試験区分の未分解残渣の乾燥重量は350mgであ
つた。同様、対照区分の乾燥重量は840mgであつ
た。
また、各々の上澄中のグルコース量は試験区分
2.1mg／100mlであり、対照区分からは検出されな
かつた。
従つて、精白生米中の澱粉のうち生澱粉分解酵
素B1018によつて約65％、500mgが溶解したこと
になる。このうち、約２％がグルコースとして検
出された。[Table] When producing raw starch degrading enzyme B1018, inoculate Bacillus sp.
By culturing with aeration for 25 to 40 hours at °C,
A significant amount of raw starch-degrading enzyme B1018 can be produced in the culture medium. As a medium, carbon sources such as glucose, maltose, starch, and cornstarch, polypeptone,
It is preferable to include a nitrogen source such as meat extract, urea, ammonia, and an inorganic nitrogen compound, and nutrients such as yeast extract and corn steep liquor as appropriate. Preferably, one or more types of raw starches are added to the medium. The cells of the resulting culture solution are separated, and since the supernatant contains raw starch-degrading enzyme B1018, the supernatant can be used as it is as an enzyme solution. In addition, the enzyme can be precipitated from the supernatant by the ammonium sulfate precipitation method and then dialyzed to obtain the crude enzyme.Furthermore, the dialyzed solution can be subjected to DEAE-cellulose column chromatography, Sephadex G75 column chromatography. Purified enzymes can also be obtained by chromatography, CM cellulose column chromatography, etc. Next, examples and usage examples of the present invention will be shown. α-amylase activity was determined using the Fuwa method [(Fuwa, J.
Biochem. 41 , 583 1954)]. Proteins were measured using the method of Lowry [OHLowry etal., J.Biol.Chem. 193 , 265
(1951)] using bovine serum albumin as a standard. Example 1 500ml of Bacillus sp.B1018, FERM P-9840
In 10 Erlenmeyer flasks, inoculate 100 ml of a medium with a composition of 1.0% glucose, 1.0% polypeptone, 0.5% raw cornstarch, 0.5% meat extract, and 0.2% yeast extract (PH 7.0), and store at 45°C for 36 hours. Cultured with shaking for hours. Centrifuge the obtained culture solution (10000 rpm, 15 minutes)
The bacterial cells were removed to obtain 820 ml of culture supernatant. Add 496g of ammonium sulfate to this culture supernatant to achieve a saturation of 0.8. After leaving the solution at 4°C, 1.13g of protein was precipitated (specific activity: 580U/mg protein).
I got it. The obtained protein was dissolved in 5mM Tris/HCl buffer (PH8.0) and dialyzed against 50mM Tris/HCl buffer (PH8.0) + 2mM calcium chloride. This dialysed fluid was subjected to DEAE-cellulose column chromatography to be adsorbed, and 50mM Tris.
Fractions eluted with hydrochloric acid (PH8.0) were collected. 56 g of ammonium sulfate (0.8 saturation) was added to 92 ml of this recovered fraction.
was added and left at 4°C for 12 hours to precipitate the protein. The obtained protein was further dialyzed against 50mM Tris/HCl buffer (PH5.6) + 2mM calcium chloride solution. The dialyzed fluid is further subjected to Sephadex G75 column chromatography. Then add 0.8 to the active fraction
Proteins were precipitated by adding ammonium sulfate to saturation. The obtained protein was dissolved in 10mM hydrochloric acid buffer (PH
5.6), dialyzed against the same buffer, applied the dialyzed solution to CM-cellulose column chromatography, adsorbed it, eluted with 10mM acetate buffer with a concentration gradient of sodium chloride (0-0.3M), and separated the active fraction. and purified raw starch degrading enzyme B1018 6.6 mg (yield 15.6
%, specific activity 15400U/mg protein). Example 2 The ammonium sulfate 0.8 saturated precipitate obtained in Example 1 was dialyzed and then subjected to DEAE-cellulose column chromatography to obtain the elution curve shown in FIG. Collect fractions No. 3 to 15 in Figure 1,
Further, the protein was precipitated with ammonium sulfate at 0.8 saturation, dialyzed, and subjected to Sephadex G75 column chromatography to obtain the elution curve shown in Figure 2. Fractions No. 25 to 45 in Figure 2 were collected and precipitated again with 0.8 saturated ammonium sulfate.
After dialysis, it was subjected to CM cellulose column chromatography to obtain the elution curve shown in Figure 3. Fractions No. 35 to 39 in FIG. 3 were collected and freeze-dried to obtain purified raw starch degrading enzyme B1018 powder. Usage example 1 1g of polished raw rice powder (77.4% starch, 14.5% moisture)
%, other 8.1%) was suspended in 500 ml of phosphate buffer, and the raw starch degrading enzyme B1018 obtained in Example 1 was added.
10 mg was added and the mixture was reacted at 45° C. for 4 hours with stirring. As a control, 1 g of raw rice powder was similarly suspended in 500 ml of 0.02M phosphate buffer and incubated at 45℃ for 4 hours with stirring.
Allowed time to react. After the reaction is complete, separate the reaction solutions from each section into test and control sections.
480 ml of the supernatant was separated into a precipitate by centrifugation at 10,000 rpm for 5 minutes. Each supernatant is Boehringer Mannheim Yamanouchi
Glucose measurement kit manufactured by Co., Ltd. (product number 716251)
The amount of glucose was measured. Precipitate at 100℃, 2
After drying for a period of time, the dry weight of the undecomposed portion was measured. The dry weight of the undecomposed residue in the test section was 350 mg. Similarly, the dry weight of the control section was 840 mg. In addition, the amount of glucose in each supernatant is determined by the test category.
It was 2.1mg/100ml and was not detected in the control section. Therefore, about 65%, or 500 mg, of the starch in the milled raw rice was dissolved by raw starch degrading enzyme B1018. Of this, about 2% was detected as glucose.
第１図は実施例２におけるDEAE−セルロース
カラムクロマトグラフイーの溶出曲線を示す図
で、第２図は同じくセフアデツクスG75カラムク
ロマトグラフイーの溶出曲線を示す図で、第３図
は同じくCMセルロースカラムクロマトグラフイ
ーの溶出曲線を示す図である。
Figure 1 is a diagram showing the elution curve of DEAE-cellulose column chromatography in Example 2, Figure 2 is a diagram showing the elution curve of SEPHADEX G75 column chromatography, and Figure 3 is a diagram showing the elution curve of DEAE-cellulose column chromatography in Example 2. It is a figure showing the elution curve of chromatography.
Claims (1)
B1018 １ 作用及び基質特異性 生澱粉を分解し、オリゴ糖、マルトース、グ
ルコースを同時に生成する。また、α−サイク
ロデキストリン、及びβ−サイクロデキストリ
ンを分解する。 ２ 至適PH及び安定PH範囲 リン酸緩衝液を用いた場合、至適PHは6.0±
0.5、安定範囲PHは4.5〜12である。 ３ 至適温度 この酵素の至適温度は55℃である。 ４ PH、温度による失活条件 PH３以下、12以上で失活する。10ｍＭ
CaCl2存在下で70℃、30分で失活する。 ５ 分子量 78000（SDS−PAGE法による）。 ２ バチルス属に属する生澱粉分解酵素B1018生
産菌を培養し、生澱粉分解酵素B1018を採取する
ことを特徴とする生澱粉分解酵素B1018の製造
法。 ３ 生澱粉分解酵素B1018を生産するバチルス
sp.B1018。[Claims] 1. Raw starch-degrading enzyme having the following physical and chemical properties:
B1018 1 Action and substrate specificity Decomposes raw starch and simultaneously produces oligosaccharides, maltose, and glucose. It also decomposes α-cyclodextrin and β-cyclodextrin. 2 Optimal PH and stable PH range When using phosphate buffer, the optimal PH is 6.0±
0.5, stable range PH is 4.5-12. 3. Optimal temperature The optimal temperature for this enzyme is 55°C. 4 Inactivation conditions due to PH and temperature Inactivation occurs at PH of 3 or lower and 12 or higher. 10mM
Inactivates in the presence of CaCl2 at 70°C for 30 minutes. 5 Molecular weight 78000 (according to SDS-PAGE method). 2. A method for producing raw starch-degrading enzyme B1018, which comprises culturing raw starch-degrading enzyme B1018-producing bacteria belonging to the genus Bacillus and collecting raw starch-degrading enzyme B1018. 3 Bacillus that produces raw starch degrading enzyme B1018
sp.B1018.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4215988A JPH01218587A (en) | 1988-02-26 | 1988-02-26 | Novel enzyme, production thereof and producing bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4215988A JPH01218587A (en) | 1988-02-26 | 1988-02-26 | Novel enzyme, production thereof and producing bacterium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01218587A JPH01218587A (en) | 1989-08-31 |
| JPH0325155B2 true JPH0325155B2 (en) | 1991-04-05 |
Family
ID=12628171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4215988A Granted JPH01218587A (en) | 1988-02-26 | 1988-02-26 | Novel enzyme, production thereof and producing bacterium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01218587A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU620412B2 (en) * | 1988-07-01 | 1992-02-20 | Showa Denko Kabushiki Kaisha | Thermostable amylase and use thereof |
-
1988
- 1988-02-26 JP JP4215988A patent/JPH01218587A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01218587A (en) | 1989-08-31 |
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