JPS63202381A - Stabilization of enzyme - Google Patents
Stabilization of enzymeInfo
- Publication number
- JPS63202381A JPS63202381A JP62034109A JP3410987A JPS63202381A JP S63202381 A JPS63202381 A JP S63202381A JP 62034109 A JP62034109 A JP 62034109A JP 3410987 A JP3410987 A JP 3410987A JP S63202381 A JPS63202381 A JP S63202381A
- Authority
- JP
- Japan
- Prior art keywords
- pullulanase
- glucoamylase
- acid
- enzyme
- dicarboxylic acid
- 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
- 108090000790 Enzymes Proteins 0.000 title description 7
- 102000004190 Enzymes Human genes 0.000 title description 7
- 230000006641 stabilisation Effects 0.000 title 1
- 238000011105 stabilization Methods 0.000 title 1
- 108090000637 alpha-Amylases Proteins 0.000 claims abstract description 35
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 4
- 230000000087 stabilizing effect Effects 0.000 claims description 4
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 abstract description 20
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 abstract description 13
- 102100022624 Glucoamylase Human genes 0.000 abstract description 12
- 239000001361 adipic acid Substances 0.000 abstract description 10
- 235000011037 adipic acid Nutrition 0.000 abstract description 10
- 244000005700 microbiome Species 0.000 abstract description 4
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 3
- 241000228245 Aspergillus niger Species 0.000 abstract description 2
- 201000008225 Klebsiella pneumonia Diseases 0.000 abstract description 2
- 241000588747 Klebsiella pneumoniae Species 0.000 abstract description 2
- 206010035717 Pneumonia klebsiella Diseases 0.000 abstract description 2
- 241000187747 Streptomyces Species 0.000 abstract description 2
- 241000588748 Klebsiella Species 0.000 abstract 1
- 229920002472 Starch Polymers 0.000 description 9
- 229960000250 adipic acid Drugs 0.000 description 9
- 235000019698 starch Nutrition 0.000 description 9
- 239000008107 starch Substances 0.000 description 9
- 125000004432 carbon atom Chemical group C* 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 108010065511 Amylases Proteins 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 4
- 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 4
- 239000002253 acid Substances 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- BDJRBEYXGGNYIS-UHFFFAOYSA-N nonanedioic acid Chemical compound OC(=O)CCCCCCCC(O)=O BDJRBEYXGGNYIS-UHFFFAOYSA-N 0.000 description 4
- WLJVNTCWHIRURA-UHFFFAOYSA-N pimelic acid Chemical compound OC(=O)CCCCCC(O)=O WLJVNTCWHIRURA-UHFFFAOYSA-N 0.000 description 4
- 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 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 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 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 239000004373 Pullulan Substances 0.000 description 2
- 229920001218 Pullulan Polymers 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229940025131 amylases Drugs 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 150000001991 dicarboxylic acids Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 235000019423 pullulan Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 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 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108010019077 beta-Amylase Proteins 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- YVSCCMNRWFOKDU-UHFFFAOYSA-N hexanedioic acid Chemical compound OC(=O)CCCCC(O)=O.OC(=O)CCCCC(O)=O YVSCCMNRWFOKDU-UHFFFAOYSA-N 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 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 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 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 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】
〔技術分野〕
本発明は、プルラナーゼの安定化方法に関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for stabilizing pullulanase.
プルラナーゼは、プルランのα−１，６−グルコシド結
合を切断し、最終的にマルトトリオースを生成する酵素
であり、これまで種々の細菌、放線菌など多種類の微生
物により生産されることが知られている。Pullulanase is an enzyme that cleaves the α-1,6-glucoside bond of pullulan and ultimately produces maltotriose, and it has been known that it is produced by many types of microorganisms, including various bacteria and actinomycetes. It is being
プルラナーゼは、アミロペクチン、澱粉、あるいはこれ
らの微生物に存在するα−１，６−グルコシド結合も分
解することができるため、α−１，６−グルコシド結合
分解能を持たないβ−アミラーゼや各種のα−１，６−
アミラーゼなどと併用して澱粉に作用させると、これら
アミラーゼを単独に作用させる場合よりも高い収量で各
アミラーゼの生産物を得ることができる０例えば、β−
アミラーゼを単独に澱粉に作用させたときのマルトース
の収量は約５５％であるが、プルラナーゼの存在下で作
用させると、約９０％の極めて高い収量でマルトースを
生産できる。Pullulanase can also decompose α-1,6-glucoside bonds present in amylopectin, starch, or these microorganisms, so it cannot be used with β-amylase or various α-amylases that do not have the ability to decompose α-1,6-glucoside bonds. 1,6-
When these amylases are used together to act on starch, the products of each amylase can be obtained in higher yields than when these amylases are used alone.For example, β-
When amylase is used alone on starch, the yield of maltose is about 55%, but when it is used in the presence of pullulanase, maltose can be produced with an extremely high yield of about 90%.
最近、プルラナーゼはグルコアミラーゼと併用すると、
グルコアミラーゼ単独の場合よりも高い収量でグルコー
スが生産できることが明らかになった（特公昭５４−２
９５７０、特公昭５７−３９、特公昭５７−１７４０８
９他）。Recently, when pullulanase is used in combination with glucoamylase,
It became clear that glucose could be produced in a higher yield than when glucoamylase was used alone (Special Publication No. 54-2
9570, Special Publication No. 57-39, Special Publication No. 57-17408
9 and others).
この場合のプルラナーゼは、グルコアミラーゼの持つα
−１，６−グルコシド結合切断能を補うことにより、澱
粉糖化反応を促進し、グルコースの増収に有効な働きを
するものと考えられている。In this case, pullulanase is α of glucoamylase.
It is believed that by supplementing the ability to cleave -1,6-glucoside bonds, it promotes the starch saccharification reaction and acts effectively in increasing the yield of glucose.
しかしながら、グルコアミラーゼは微生物起源によって
も、若干、異なるが一般的にはｐＨ４〜５、温度５５〜
６０℃の範囲において、最も効率的に作用する酵素であ
る。これに対し、これまで知られている多くのプルラナ
ーゼは、ｐＨ６〜７、温度５０〜５５℃に最適作用域が
あるため、グルコアミラーゼの作用域と一致しない、こ
のため、いずれかの酵素活性を犠牲にして反応しなけれ
ばならなかった。However, glucoamylase generally has a pH of 4-5 and a temperature of 55-55, although it varies slightly depending on the microbial origin.
It is an enzyme that works most efficiently in the 60°C range. On the other hand, many of the pullulanases known so far have an optimal action range at pH 6-7 and temperature 50-55°C, which does not match the action range of glucoamylase. I had to sacrifice and react.
本発明者らは、グルコアミラーゼの好適作用域で、プル
ラナーゼを作用させるためのプルラナーゼの安定化方法
について、種々、検討を加えてきた結果、プルラナーゼ
を炭素数５以上１０以下のジカルボン酸、特に炭素数６
個のアジピン酸（ヘキサンジオイック アシド）で処理
すると、プルラナ−ゼが顕著に安定されることを認めた
０本発明は、この知見に基づいてなされたものである。The present inventors have conducted various studies on methods for stabilizing pullulanase to allow pullulanase to act in the preferred action range of glucoamylase. As a result, the present inventors have determined that pullulanase is a dicarboxylic acid having 5 to 10 carbon atoms, particularly carbon Number 6
It was recognized that pullulanase was significantly stabilized when treated with adipic acid (hexanedioic acid).The present invention was made based on this finding.
すなわち、本発明はプルラナーゼをジカルボン酸で処理
することを特徴′どするプルラナーゼの安定化法に関す
るものである。That is, the present invention relates to a method for stabilizing pullulanase, which is characterized by treating pullulanase with a dicarboxylic acid.
以下に、本発明の内容を更に具体的に説明する。The contents of the present invention will be explained in more detail below.
本発明を、例えばグルコアミラーゼと併用するプルラナ
ーゼに通用する場合、プルラナーゼとしては、少なくと
も最適温度が５０℃以上（１％プルラン濃度） 、ｐｕ
ｓにおいても作用することが要求される。このような酵
素は、バシルス属、クレプシラ（ニーロバフタ）属、ス
トレプトマイセス属など種々の微生物により生産され、
例えば、クレブシラ・ニューモニア（Ｋｌｅｂｓｉｌｌ
ａ ｐｎｅｕｍｏｎｉａ）のプルラナーゼやバシルス・
ズプチルス（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）のプ
ラナーゼが使用される。For example, when the present invention is applicable to pullulanase used in combination with glucoamylase, the optimum temperature for pullulanase is at least 50°C or higher (1% pullulan concentration), pu
It is also required to act on s. Such enzymes are produced by various microorganisms such as Bacillus, Klepsilla, and Streptomyces.
For example, Klebscilla pneumonia
a pneumonia) pullulanase and Bacillus.
Bacillus subtilis planase is used.
本発明において使用されるジカルボン酸は、例えば、ア
ジピン酸（炭素数６）、ピメリン酸（炭素数７）、スペ
リン酸（炭素数８）、アゼライン酸（炭素数９）、ゼバ
シン酸（炭素数１０）など、一般式ＣｎＨｘｎ（Ｃｏｏ
Ｈ）ｚで表される飽和ジカルボン酸であり、炭素数とし
て５〜１０程度のものが有効であるが、特に、アジピン
酸は効果的である。Examples of dicarboxylic acids used in the present invention include adipic acid (6 carbon atoms), pimelic acid (7 carbon atoms), superric acid (8 carbon atoms), azelaic acid (9 carbon atoms), and zebacic acid (10 carbon atoms). ), etc., the general formula CnHxn(Coo
It is a saturated dicarboxylic acid represented by H)z, and those having about 5 to 10 carbon atoms are effective, and adipic acid is particularly effective.
プルラナーゼのアジピン酸による処理は、通常該酵素１
単位当り０．５〜５ミリモル量のジカルボン酸を加えて
、あらかじめプルラナーゼ剤を処理するか、あるいは該
酵素反応液中に添加される。Treatment of pullulanase with adipic acid usually results in the enzyme 1
A dicarboxylic acid in an amount of 0.5 to 5 mmol per unit is added to pre-treat the pullulanase agent or added to the enzyme reaction solution.
このような処理をされたプルラナーゼ剤はグルコアミラ
ーゼ、特にアスペルギルス・ニガー（Ａｓｐｅｒｇｉｌ
ｌｕｓ ｎｉｇｅｒ）のグルコアミラーゼの好適な作
用条件である、ｐＨ４，３〜４，５、温度５７〜６０℃
において有効に作用し、無処理のプルラナーゼに比ペブ
ルコースを０．５〜３％増収することができる。Such treated pullulanase agents contain glucoamylase, especially Aspergillus niger.
lus niger) glucoamylase, pH 4.3 to 4.5 and temperature 57 to 60°C.
It can increase the yield of pebulose by 0.5 to 3% compared to untreated pullulanase.
次に、実施例により、本発明の詳細な説明する。Next, the present invention will be explained in detail with reference to Examples.
〈実施例 ｌ〉
可溶性澱粉２％、尿素０．３５％、ＸＪＰＯｅ ０．０
５％Ｍｇ５Ｏａ ’ ＩＨｚＯＯ，０５％、にｃ ｚ
ｏ、ｓ％、ＭｎＣＪｚ５Ｘ１０−’Ｍ、ＣａＣＪ ｇｌ
Ｘｌｏ−’Ｍからなる培地５０ｍ Ｉｔを２００ｍ ｊ
！容三角フラスコに入れ、常法により殺菌後、クレブシ
ラ・ニューモニア（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕ
ｍｏｎｉａ＠） ＦＥＲＭＰ−７３８７を接種し、３０
℃で２日間振盪培養した。培養後、遠心分離して得た上
澄のプルラナーゼ活性は、培地１ｗｊ！当たり６．０単
位であった。<Example l> Soluble starch 2%, urea 0.35%, XJPOe 0.0
5%Mg5Oa 'IHzOO,05%, c z
o, s%, MnCJz5X10-'M, CaCJ gl
200 m j of 50 m It of medium consisting of Xlo-'M
! Pour into an Erlenmeyer flask, sterilize it by a conventional method, and then kill Klebsiella pneumonia.
monia@) Inoculated with FERMP-7387, 30
The cells were cultured with shaking at ℃ for 2 days. After culturing, the pullulanase activity of the supernatant obtained by centrifugation was 1wj! It was 6.0 units per unit.
該プルラナーゼ１単位当たり、アジピン酸を２ミリモル
量加え、室温で３０分間処理した後、糖化反応用酵素と
して使用した。Adipic acid was added in an amount of 2 mmol per unit of pullulanase, treated at room temperature for 30 minutes, and then used as an enzyme for saccharification reaction.
市販デキストリン（参松工業株式会社製 粉末水飴Ｓ
Ｌ Ｄ） ３．８ｇ、グルコアミラーゼ（デンマーク
ノボ社製アミログルコシダーゼＡ Ｍ Ｃ３００）２．
２Ｘ１０−”ｍＡ！、塩化カルシウムＩ Ｘｌ０−”Ｍ
、上記のアジピン酸処理、または無処理のプルラナーゼ
を澱粉ｇ当たり０６７５単位を加え、水で全量１０ｍ
ｊ！とじ、ｐｔｔａ、ｓ、温度６０℃で反応を行った。Commercially available dextrin (Sanmatsu Kogyo Co., Ltd. powder starch syrup S)
L D) 3.8g, glucoamylase (Denmark)
Amyloglucosidase A MC300 (manufactured by Novo) 2.
2X10-”mA!, Calcium Chloride I Xl0-”M
, add 0675 units of the above adipic acid-treated or untreated pullulanase per g of starch, and dilute the total amount with water to 10 m
j! The reaction was carried out at a temperature of 60°C.
反応開始後、５５時間目におけるグルコースの収量を高
速液体クロマトグラフで分析した結果は第１表に示す通
りであった。The yield of glucose 55 hours after the start of the reaction was analyzed by high performance liquid chromatography, and the results are shown in Table 1.
く以下余白〉
第１表
表から明らかなように、アジピン酸処理したプルラナー
ゼは無処理のものに比ベグルコースの増収に対し顕著な
効果を示した。As is clear from Table 1, the pullulanase treated with adipic acid had a remarkable effect on increasing the yield of beglucose compared to the untreated pullulanase.
〈実施例 ２〉
大豆粕５％、コーン・ステイープ・リカー０．６％肉エ
キス０．４％、リン酸二カリ０．３％、硫酸マグネシウ
ム（７水塩）０．１％、可溶性澱粉２％、尿素０．３％
、ソデイウム・ドデシル・サルフェート０．１％、硫ｅ
ＩＩ銅５ ｘｌＯ−’Ｍ、硫酸亜鉛Ｉ Ｘｌ０−’Ｍ、
硫ｐ鉄Ｉ Ｘ１０−’Ｍからなる培地（ｐＨ７，２）
３０ｍｆを２０ｈ ｌ容三角フラスコに入れ、常法によ
り殺菌後、バシルス・ズブチルスＴＬＩ （Ｂａｃｉｌ
ｌｕｓ ５ｕｂｔｉｌｉｓ ＴＵ）（ＦＥＲＭ Ｂ
Ｐ６８４）を接種し、３０℃で４日間培養した。培養後
、遠心分離して得た上澄中のプルラナーゼ活性は、培地
１ ｍ １１０．５単位であった。<Example 2> Soybean meal 5%, corn steep liquor 0.6%, meat extract 0.4%, dipotassium phosphate 0.3%, magnesium sulfate (heptahydrate) 0.1%, soluble starch 2 %, urea 0.3%
, sodium dodecyl sulfate 0.1%, sulfur e
II copper 5 xlO-'M, zinc sulfate I Xl0-'M,
Medium consisting of p-iron sulfate I X10-'M (pH 7.2)
Pour 30 mf into a 20 h L Erlenmeyer flask, sterilize it by a conventional method, and then add Bacillus subtilis TLI.
lus 5ubtilis TU) (FERM B
P684) was inoculated and cultured at 30°C for 4 days. After culturing, the pullulanase activity in the supernatant obtained by centrifugation was 110.5 units per 1 m of medium.
該プルラナーゼ１単位当り、アジピン酸、ピメリン酸、
スペリン酸、アゼライン酸、ゼバシン酸を各４ミリモル
量添加し、室温で３０分間処理した後、糖化に使用した
。per unit of pullulanase, adipic acid, pimelic acid,
Speric acid, azelaic acid, and zebacic acid were added in amounts of 4 mmol each, treated at room temperature for 30 minutes, and then used for saccharification.
澱粉糖化反応は、上記プルラナーゼを用いる以外、実施
例１に記載の方法と同じ組成で、ｐＨ４，４６０℃で反
応を行った。The starch saccharification reaction was carried out at pH 4, 460° C. using the same composition as in Example 1 except for using the above-mentioned pullulanase.
糖化開始後５３時間目におけるグルコースの収量を高速
液体クロマトグラフ法により測定した結果は、第２表に
示す通りであった。The yield of glucose measured 53 hours after the start of saccharification by high performance liquid chromatography was as shown in Table 2.
く以下余白〉
第２表
表から明らかなように、各ジカルボン酸処理したものは
、無処理のものに比べ有効性が認められ、特にアジピン
酸処理したものは顕著な効果を示した。As is clear from Table 2, those treated with each dicarboxylic acid were found to be more effective than those without treatment, and those treated with adipic acid showed particularly remarkable effects.
〈実施例 ３〉
実施例２で調製した酵素液各１単位に、アジピン酸をそ
れぞれ１．２．３．４ミリモル量添加し、室温で３０分
間処理後、実施例１と同様の組成、ｐｔ＋４．４．６０
℃で２日間反応を行った。得られた糖化液の糖組成は第
３表に示す通りであった。<Example 3> Adipic acid was added in an amount of 1, 2, and 3.4 mmol to each unit of the enzyme solution prepared in Example 2, and after treatment at room temperature for 30 minutes, the same composition as in Example 1, pt+4 .4.60
The reaction was carried out at ℃ for 2 days. The sugar composition of the obtained saccharified liquid was as shown in Table 3.
〈以下余白〉<Margin below>
Claims (1)
るプルラナーゼの安定化方法。A method for stabilizing pullulanase, which comprises treating pullulanase with a dicarboxylic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62034109A JPS63202381A (en) | 1987-02-17 | 1987-02-17 | Stabilization of enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62034109A JPS63202381A (en) | 1987-02-17 | 1987-02-17 | Stabilization of enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63202381A true JPS63202381A (en) | 1988-08-22 |
| JPH0236234B2 JPH0236234B2 (en) | 1990-08-16 |
Family
ID=12405110
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62034109A Granted JPS63202381A (en) | 1987-02-17 | 1987-02-17 | Stabilization of enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63202381A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5627075A (en) * | 1993-06-21 | 1997-05-06 | Boehringer Mannheim Corporation | Stable diagnostic reagent |
-
1987
- 1987-02-17 JP JP62034109A patent/JPS63202381A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5627075A (en) * | 1993-06-21 | 1997-05-06 | Boehringer Mannheim Corporation | Stable diagnostic reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0236234B2 (en) | 1990-08-16 |
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