JPH04169188A - Production and purification of alginic acid oligosaccharide - Google Patents
Production and purification of alginic acid oligosaccharideInfo
- Publication number
- JPH04169188A JPH04169188A JP2294897A JP29489790A JPH04169188A JP H04169188 A JPH04169188 A JP H04169188A JP 2294897 A JP2294897 A JP 2294897A JP 29489790 A JP29489790 A JP 29489790A JP H04169188 A JPH04169188 A JP H04169188A
- Authority
- JP
- Japan
- Prior art keywords
- alginate
- alginic acid
- produced
- oligosaccharide
- alteromonas
- 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
- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 48
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 48
- 239000000783 alginic acid Substances 0.000 title claims abstract description 23
- 229960001126 alginic acid Drugs 0.000 title claims abstract description 23
- 229920001542 oligosaccharide Polymers 0.000 title claims description 20
- -1 alginic acid oligosaccharide Chemical class 0.000 title claims description 17
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 238000000746 purification Methods 0.000 title description 2
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 244000005700 microbiome Species 0.000 claims abstract description 14
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000661 sodium alginate Substances 0.000 claims abstract description 11
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 11
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 11
- 241000590031 Alteromonas Species 0.000 claims abstract description 6
- 241000589563 Alteromonas sp. Species 0.000 claims abstract description 3
- 229940072056 alginate Drugs 0.000 claims description 25
- 230000000694 effects Effects 0.000 claims description 20
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 15
- 150000003839 salts Chemical class 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- 238000005194 fractionation Methods 0.000 claims description 3
- 238000002523 gelfiltration Methods 0.000 claims description 3
- 150000002482 oligosaccharides Chemical class 0.000 claims description 3
- 108090000856 Lyases Proteins 0.000 abstract description 5
- 102000004317 Lyases Human genes 0.000 abstract description 5
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 15
- 150000004781 alginic acids Chemical class 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000000354 decomposition reaction Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- IAJILQKETJEXLJ-SQOUGZDYSA-N L-guluronic acid Chemical compound O=C[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O IAJILQKETJEXLJ-SQOUGZDYSA-N 0.000 description 1
- 241000239205 Merostomata Species 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 241000199919 Phaeophyceae Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- GLMQHZPGHAPYIO-UHFFFAOYSA-L azanium;2-hydroxypropane-1,2,3-tricarboxylate;iron(2+) Chemical compound [NH4+].[Fe+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O GLMQHZPGHAPYIO-UHFFFAOYSA-L 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- AEMOLEFTQBMNLQ-YBSDWZGDSA-N d-mannuronic acid Chemical compound O[C@@H]1O[C@@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-YBSDWZGDSA-N 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000002196 fr. b Anatomy 0.000 description 1
- 210000003918 fraction a Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 235000000011 iron ammonium citrate Nutrition 0.000 description 1
- 239000004313 iron ammonium citrate Substances 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
- 239000013028 medium composition Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 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)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、アルギン酸オリゴ糖の製造方法及び精製法、
詳しくは微生物の生産する新規な酵素を用いてアルギン
酸ナトリウムを分解させる方法及びその分解物の精製法
に関するものである。[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for producing and purifying alginate oligosaccharide,
Specifically, the present invention relates to a method for decomposing sodium alginate using a novel enzyme produced by microorganisms, and a method for purifying the decomposed product.
［従来の技術］
褐藻類の主要な細胞間粘質多糖類であるアルギン酸は、
その主構成分子としてＤ−マンヌロン酸及びＬ−グルロ
ン酸を含むもので、このアルギン酸を低分子化すれば、
アルギン酸オリゴ糖を得ることができると考えられる。[Prior art] Alginic acid, the main intercellular mucilage polysaccharide of brown algae, is
It contains D-mannuronic acid and L-guluronic acid as its main constituent molecules, and if this alginic acid is reduced in molecular weight,
It is believed that alginate oligosaccharides can be obtained.
しかしながら、アルギン酸の水？８液は粘調性が高く、
該アルギン酸は、該水ＮＯ中のカルシウム等と金属塩を
つくりゲル化するため、非常に分解され難いことが知ら
れている。However, alginate water? 8 liquid has high viscosity,
It is known that alginic acid is extremely difficult to decompose because it forms a gel with calcium and the like in the water NO.
そこで、アルギン酸の処理においては、いくつかの微生
物を用いて分解させる試みがなされている０例えば、特
開昭６３−２１４１９２号公報には、アルテロモナス属
に属する微生物の生成するアルギン酸リアーゼを用いて
アルギン酸ナトリウムを分解し、アルギン酸オリゴ環を
製造する方法が開示されている。Therefore, in the treatment of alginic acid, attempts have been made to decompose it using several microorganisms. A method for decomposing sodium and producing alginate oligocycles is disclosed.
また、上記のような方法で製造したアルギン酸オリゴ環
からの塩類等の低分子物質の除去は、アルギン酸オリゴ
糖自体も電荷を有しているため一般のイオン交換樹脂に
よる処理では困難であった。In addition, it has been difficult to remove low-molecular substances such as salts from the alginic acid oligosaccharides produced by the method described above by treatment with a general ion exchange resin because the alginic acid oligosaccharides themselves have electric charges.
上記公報に記載のアルギン酸オリゴ環の製造方法におい
ては、そこで用いられる上記のアルギン酸リアーゼは、
その酵素活性の至適温度が４０℃でありこの温度を超え
ると失活し易いため、室温では取り扱い難く、酵素反応
の温度を高めて他の微生物の混在を防ぐ上でも耐熱性が
充分とは言い難かった。In the method for producing alginate oligocycles described in the above publication, the above alginate lyase used therein is
The optimum temperature for the enzyme's activity is 40°C, and if it exceeds this temperature, it is likely to be inactivated, so it is difficult to handle at room temperature, and its heat resistance is insufficient to raise the temperature of the enzyme reaction and prevent the contamination of other microorganisms. It was hard to say.
従って、本発明の目的は、室温で取扱いが容易で、且つ
酵素活性の至適温度及び熱安定領域が他の微生物の混在
を防ぐに足る充分高い酵素で、しかも工業的に容易に生
産できる酵素を用いるアルギン酸オリゴ環の製造方法を
提供することにある。Therefore, an object of the present invention is to develop an enzyme that is easy to handle at room temperature, has an optimal temperature for enzyme activity and a thermostability range high enough to prevent the contamination of other microorganisms, and can be easily produced industrially. An object of the present invention is to provide a method for producing an alginate oligocycle using the method.
また、本発明の別の目的は、製造されたアルギン酸オリ
ゴ環から、塩類等の低分子物質を容易に除去し得るアル
ギン酸オリゴ環の精製法を提供することにある。Another object of the present invention is to provide a method for purifying alginic acid oligo rings that can easily remove low-molecular substances such as salts from the produced alginic acid oligo rings.
（課題を解決するための手段）
本発明者等は種々検討を行った結果、魚介類の腸及びそ
の内容物よりアルギン酸ナトリウムを唯一の炭素源とし
てスクリーニングを実施し、カブトガニの腸より分離し
た微生物（菌株）の生産する酵素であるアルギン酸リア
ーゼを用いることにより、上記目的を達成し得ることを
知見すると共に、特定の担体を用いることにより、アル
ギン酸オリゴ環を効率良く精製できることを知見した。(Means for Solving the Problem) As a result of various studies, the present inventors screened the intestines of seafood and their contents using sodium alginate as the only carbon source, and the microorganisms isolated from the intestines of horseshoe crabs were screened. We have found that the above objectives can be achieved by using alginate lyase, an enzyme produced by A.
本発明は、上記知見に基づいてなされたもので、アルギ
ン酸ナトリウムからアルギン酸オリゴ環を製造するに際
し、アルテロモナス属に属する微生物によって生産され
る、酵素活性の至適温度が４５〜５５゛Ｃであるアルギ
ン酸リアーゼを用いることを特徴とするアルギン酸オリ
ゴ環の製造方法を提供するものである。The present invention was made based on the above-mentioned findings, and uses alginic acid, which is produced by a microorganism belonging to the genus Alteromonas and whose optimal temperature for enzyme activity is 45 to 55°C, when producing alginate oligocycles from sodium alginate. The present invention provides a method for producing an alginate oligocycle characterized by using lyase.
また、本発明は、上記製造方法により製造されるアルギ
ン酸オリゴ環の好ましい精製法として、アルギン酸オリ
ゴ環をオリゴ垢分画ゲル′ａ過担体（分画範囲１００〜
１，８００ダルトン）と接触させて該アルギン酸オリゴ
槽中に含まれる塩類等の低分子物質を除去することを特
徴とするアルギン酸オリゴ槽の精製法を提供するもので
ある。In addition, the present invention provides a preferred method for purifying the alginate oligo-ring produced by the above-mentioned production method, in which the alginate oligo-ring is purified using an oligo-scale fractionation gel 'a supercarrier (fraction range 100~
1,800 daltons) to remove low-molecular substances such as salts contained in the alginic acid oligo tank.
以下に、本発明について詳述する。The present invention will be explained in detail below.
先ず、本発明のアルギン酸オリゴ槽の製造方法で用いる
微生物（本菌株）について説明すると、その形態学的性
質及び生理学的性質は下記の第１表に示す通りである。First, the microorganism (this strain) used in the method for producing an alginate oligo bath of the present invention will be explained. Its morphological properties and physiological properties are as shown in Table 1 below.
第１表 菌株の性質
・形態学的性質
■）グラム染色性 −−−−−−一一一・−陰性２）細
胞の形状−・−・・＝−桿菌
３）コロニーの色調 ・−−−−−・−乳白色４）運動
性の有無 −−−−一一−−−−−− あり５）鞭毛の
有無−・−−一一一−−・・−極鞭毛・生理学的性質
１）Ｏ−Ｆテスト −酸化型
２）オキンダーゼテスト 陽性３）ゼラチンの
分解 −・ −陽性
４）ＤＮＡの分解 −９，−陽性
５）好 塩 性 −−ｍ−陽性
ＧＣ含量−−−−４９，１ｍｏ１％
上記第１表に示す菌株の性質に基づいて清水らの方法（
海洋微生物研究法、学会出版センター、２２８〜２３９
（１９８５））に従って同定を試みた結果、上記微生
物（本菌株）は、アルテロモナス属に属するものである
ことが判明した。本菌株は微生物工業技術研究所に、微
工研菌寄第１１６８５号として寄託されている。Table 1 Characteristics and Morphological Properties of Bacterial Strains■) Gram staining property -------111・-Negative 2) Cell shape---=-Bacillus 3) Colony color tone ・---- ---・- Milky white 4) Motility --- 11 --- Yes 5) Flagella present ---- 111 --- Polar flagella/Physiological properties 1) O - F test - Oxidized type 2) Okindase test Positive 3) Decomposition of gelatin - -Positive 4) Decomposition of DNA -9, -Positive 5) Halophilicity -m-Positive GC content---49, 1mol1% Based on the properties of the strains shown in Table 1 above, the method of Shimizu et al.
Marine Microbial Research Methods, Society Publishing Center, 228-239
(1985)), it was found that the above microorganism (this strain) belonged to the genus Alteromonas. This strain has been deposited with the Microbial Technology Research Institute as Microbiological Research Institute No. 11685.
而して、本発明で用いられるアルギン酸リアーゼは、上
記第１表に示す性質を有する菌株を、実施例１として示
す後記の〔培養法〕等により培養して得られるもので、
その酵素的性質は次の通りである。Therefore, the alginate lyase used in the present invention is obtained by culturing a strain having the properties shown in Table 1 above by the [Culture method] described below as Example 1, etc.
Its enzymatic properties are as follows.
（１）作用ニアルギン酸を基質としてアルギン酸リアー
ゼを反応させた時、反応生成物であるアルギン酸オリゴ
糖の二重結合に由来する特異吸収波長である２３０ ｎ
ｍ における吸光度の増加、及び生じるオリゴ垢によ
る還元力の増加が確認された。(1) Effect When alginate lyase is reacted with nialginic acid as a substrate, a specific absorption wavelength of 230 n is derived from the double bond of alginate oligosaccharide, which is the reaction product.
An increase in absorbance at m and an increase in reducing power due to the oligoscum produced were confirmed.
（２）至適ｐ１１本発明で用いるアルギン酸リアーゼの
各ｐＨにおける相対活性量を示す第１図のグラフから明
らかなように、ｐＨ７，０〜７．５の範囲で相対活性量
が高く相対活性量が最大になるｐＨ７，０が至適ｐＨで
ある。(2) Optimal p11 As is clear from the graph in Figure 1 showing the relative activity at each pH of alginate lyase used in the present invention, the relative activity is high in the pH range of 7.0 to 7.5. The optimum pH is pH 7.0, at which the maximum value is obtained.
（３）至適温度及び熱安定性ニアルギン酸リアーゼの各
温度における相対活性量を示す第２図のグラフから明ら
かなように、相対活性量が最大となる５０’Ｃが至適温
度であり、また、アルギン酸リアーゼの各温度における
２０分間の熱処理による熱安定性を示す第３図のグラフ
から明らかなように、熱安定性は、粗酵素で５０℃付近
まで安定であり、室温ｉｆｆ縮を行っても酵素活性の低
下は認められなかった。尚、本発明では、酵素活性の至
適温度が４５〜５５℃であるアルギン酸リアーゼを用い
るため、アルギン酸ナトリウムとアルギン酸リアーゼと
の反応は４５〜５５“Ｃで行うのが好ましい。(3) Optimal temperature and thermostability As is clear from the graph in FIG. 2 showing the relative activity of nialginate lyase at each temperature, the optimal temperature is 50'C, where the relative activity is maximum, In addition, as is clear from the graph in Figure 3 showing the thermal stability of alginate lyase after heat treatment for 20 minutes at various temperatures, the thermal stability of the crude enzyme is stable up to around 50°C; However, no decrease in enzyme activity was observed. In the present invention, since alginate lyase whose optimum temperature for enzyme activity is 45 to 55<0>C is used, the reaction between sodium alginate and alginate lyase is preferably carried out at 45 to 55<0>C.
（４）酵素活性：下記組成の反応液を用い、アルギン酸
ナトリウムとアルギン酸リアーゼとを５０℃にて１０分
間反応させ、生成したオリゴ糖量をネルソン・ソモギー
法により測定することにより、アルギン酸リアーゼの酵
素活性を測定できる。この酵素活性は、１μ５ｏｌｅの
マンニュロン酸に相当するアルギン酸オリゴ糖を生成す
る酵素量を１単位として示す。(4) Enzyme activity: Using a reaction solution with the following composition, sodium alginate and alginate lyase were reacted at 50°C for 10 minutes, and the amount of oligosaccharide produced was measured by the Nelson-Somogyi method. Activity can be measured. This enzyme activity is expressed as one unit, which is the amount of enzyme that produces alginate oligosaccharide corresponding to 1 μ5 ole of mannuronic acid.
（反応液の組成）
・０．５％アルギン酸ナトリウム（和光純薬製）を含む
０．０５ Ｍリン酸ナトリウム緩衝液（ｐＨ７，０）
−−−−−−−−−−−−−−−−−−−−−−−−０
，４５ｍ・酵素液 −−−−−−−−−−−−−−−−
−−−−−−−−０，０５ｄ尚、本発明においては、本
菌株を通常の変異手段を適用して得られる変異株であっ
てアルギン酸リアーゼ産生能を有する菌株を培養して得
られるアルギン酸リアーゼも使用することもできる。(Composition of reaction solution) - 0.05 M sodium phosphate buffer (pH 7.0) containing 0.5% sodium alginate (manufactured by Wako Pure Chemical Industries)
−−−−−−−−−−−−−−−−−−−−−−−−0
, 45m・Enzyme solution −−−−−−−−−−−−−−−−
----------0.05dIn the present invention, alginate lyase produced by culturing a strain that is a mutant strain obtained by applying ordinary mutation means to this strain and has the ability to produce alginate lyase is used. Lyases can also be used.
実施例１は本発明で用いるアルギン酸リアーゼを得るた
めの前記微生物（本菌株）の培養法を示し、実施例２は
本発明のアルギン酸オリゴ糖の製造方法の実施例を示し
、実施例３は本発明のアルギン酸オリゴ糖の精製法の実
施例を示す。Example 1 shows a method for culturing the microorganism (this strain) to obtain alginate lyase used in the present invention, Example 2 shows an example of the method for producing alginate oligosaccharides of the present invention, and Example 3 shows a method for producing alginate oligosaccharides of the present invention. An example of the method for purifying alginate oligosaccharides of the invention is shown.
実施例１
〔培地組成〕
・アルギン酸ナトリウム −−−−−ｌｏｇ・人工渇
水 −−−−−＝−−−−１０００ｒｎｌＦｅＳ
ｔＯＣｋ−１ｍ！
・ＰｌｓＬｏｃｋ−−−一−−−−−−２ｒＩ１１・Ｎ
Ｈ４５Ｌｏｃｋ−−−−一−−５１ｄ−１？Ｉ トＩ
Ｊ スー塩ａ［衝′ｅ（Ｐ Ｈ７，８） −５０ａＮ人
工海水：塩化ナトリウム３００Ｉ＠Ｍ、塩化カリウム１
０ｍＭ、硫酸マグネシウム（７水和物）５０１１Ｍ、塩
化カルシウム（２水塩） １０＋ａＭＦｅ ５ｔｏｃｋ
：クエン酸鉄アンモニウム１０ｇ／ｌｏｏｓＭ脱塩水
Ｐｉ ５ｔｏｃｋ−リン酸水素二カリウム（３水和物）
７．５ｇ／１００ｍ脱塩水
ＮＨａ ５ｔｏｃｋ：塩化アンモニウム２０ｇ／１００
ＩＩｉ脱塩水上記組成の培地を用い、凍結乾燥保存菌体
アルテロモナス・エスピーに１７８６株を２回前培養（
２ｏ ’ｃ、１日）後、本培養（２５℃５１日）を行っ
た。その結果、酵素活性が培養液１ｙｄ当り０．８４単
位であるアルギン酸リアーゼ培養液が生産された。Example 1 [Medium composition] ・Sodium alginate ------log ・Artificial drought ------=----1000rnlFeS
tOCk-1m!・PlsLock---1---2rI11・N
H45Lock----1--51d-1? I
J Sioux salt a [op'e (PH7,8) -50aN Artificial seawater: Sodium chloride 300I@M, potassium chloride 1
0mM, magnesium sulfate (heptahydrate) 5011M, calcium chloride (dihydrate) 10+aMFe 5tock
: Iron ammonium citrate 10g/loosM demineralized water Pi 5tock-dipotassium hydrogen phosphate (trihydrate)
7.5g/100m Demineralized water NHa 5tock: Ammonium chloride 20g/100
IIi Desalinated water Using a medium with the above composition, preculture 1786 strain twice on the freeze-dried preserved bacterial cell Alteromonas sp.
2o'c, 1 day), main culture (25°C, 51 days) was performed. As a result, an alginate lyase culture solution with an enzyme activity of 0.84 units per yd of culture solution was produced.
上記培養液を、ブレースジャパン社のアミコンの限外濾
過膜で濃縮して、分子量１００００以下の物質を除去し
アルギン酸リアーゼ濃縮液とした。The above culture solution was concentrated using an Amicon ultrafiltration membrane manufactured by Brace Japan to remove substances with a molecular weight of 10,000 or less to obtain an alginate lyase concentrate.
実施例２
アルギン酸ナトリウム（４２，３ｇ）を１６００ｄの０
．０５ Ｍリン酸ナトリウム緩衝液（ｐＨ７，０＞に熔
解後、これに実施例１で得られたアルギン酸リアーゼ濃
縮液（３９７，５Ｕ）を加え、５０’Ｃで２４時間撹拌
しながら反応させ、酸性基（酢酸基）を有するアルギン
酸オリゴ糖の混合物を得た。この混合物中のアルギン酸
オリゴ糖の重合度は３〜５が主であった。Example 2 Sodium alginate (42.3 g) was added to 1600 d of 0
．． After dissolving to 05 M sodium phosphate buffer (pH 7.0), the alginate lyase concentrate (397.5 U) obtained in Example 1 was added thereto, and the mixture was reacted with stirring at 50'C for 24 hours to give an acidic A mixture of alginic acid oligosaccharides having groups (acetic acid groups) was obtained.The degree of polymerization of the alginic acid oligosaccharides in this mixture was mainly 3 to 5.
実施例３
実施例２で製造したオリゴ糖をＢｌｏ−Ｇｅ１ Ｐ−２
（日本バイオランド ラボラトリーズ■の商品名）とい
う担体〔オリゴ糟分画（分画範囲１００〜１８００ダル
トン）ゲル濾過担体〕を充填したカラムを通過させた。Example 3 The oligosaccharide produced in Example 2 was used as Blo-Ge1 P-2
It was passed through a column filled with a carrier called (trade name of Nippon Bioland Laboratories ■) [oligo fractionation (fraction range 100-1800 daltons) gel filtration carrier].
その結果、アルギン酸オリゴ環に対してはイオン排除と
いう現象が生して、アルギン酸分解物のほとんどは担体
から排除されることが明らかになった。すなわち、この
ことについて第４図により説明すると、アルギン酸オリ
ゴ環をｐＨの低い緩衝液、例えば、０．１ Ｍ酢酸緩衝
液（ｐＨ４，０）で溶出すると、イオン排除が生ぜず、
アルギン酸オリゴ環はフラクション番号２０〜７００間
に分画される。これに対して、脱塩水で溶出すると、イ
オン排除が生し、アルギン酸オリゴ１１！（画分Ａ）は
フラクション番号１０のあたりに溶出し塩類（画分Ｂ）
はフラクション番号８０あたりに溶出する。As a result, it was revealed that a phenomenon called ion exclusion occurs in the alginic acid oligo ring, and most of the alginic acid decomposition products are excluded from the carrier. That is, to explain this with reference to FIG. 4, when alginate oligo rings are eluted with a low pH buffer, for example, 0.1 M acetate buffer (pH 4.0), ion exclusion does not occur;
Alginic acid oligo rings are fractionated between fraction numbers 20-700. On the other hand, when eluted with demineralized water, ion exclusion occurs and alginate oligo 11! (Fraction A) is eluted around fraction number 10 and salts (Fraction B)
is eluted around fraction number 80.
従って、この現象を利用して、アルギン酸分解物からＮ
ａＣ１や緩衝液に用いた塩などの低分子物質の除去が可
能となった。Therefore, by utilizing this phenomenon, N
It has become possible to remove low molecular weight substances such as aC1 and salts used in buffer solutions.
１） 本発明のアルギン酸オリゴ環の製造方法によれば
、酵素として、室温で取扱いが容易で、且つ酵素活性の
至適温度及び熱安定頭載が他のＶ＆住物の混在を防ぐに
足る充分高い酵素で、しかも工業的に容易に生産できる
酵素を用いているため、極めて効率良くアルギン酸オリ
ゴ環を製造できる。1) According to the method for producing an alginate oligocyclic ring of the present invention, it is easy to handle as an enzyme at room temperature, and the optimum temperature for enzyme activity and thermal stability are sufficient to prevent the mixing of other V&Ns. Since an enzyme that is highly efficient and can be easily produced industrially is used, alginate oligocycles can be produced extremely efficiently.
２） また、従来電荷を有するオリゴ環（例えばアルギ
ン酸分解物）より脱塩を行うことは不可能に近かったが
、本発明の精製法によれば、アルギン酸オリゴ環からの
脱塩処理が可能になった（例えば、アルギン酸分解物に
ある特定の金属を結合させた後、過剰な試薬類を除去す
ることも可能である。）。2) In addition, conventionally it was nearly impossible to desalt from charged oligo rings (e.g., alginic acid decomposition products), but according to the purification method of the present invention, it is now possible to desalt from alginic acid oligo rings. (For example, it is also possible to remove excess reagents after binding a certain metal to an alginic acid decomposition product.)
第１図は本発明で用いるアルギン酸リアーゼの各ｐＨに
おける相対活性量を示すグラフ、第２図はアルギン酸リ
アーゼの各温度における相対活性量を示すグラフ、第３
図はアルギン酸リアーゼの各温度における２０分間処理
による熱安定性を示すグラフ、第４図はアルギン酸オリ
ゴ環を前記ゲル濾過担体を充填したカラムを通過させ、
脱塩水で溶出した時の溶出パターンを示し、グラフＡは
アルギン酸オリゴ環の溶出パターンを示し、グラフＢは
塩の溶出パターンを示す。
第１図
ｐＨ
第２図
ΔＬｌ″ＣノFigure 1 is a graph showing the relative activity of alginate lyase used in the present invention at each pH, Figure 2 is a graph showing the relative activity of alginate lyase at each temperature, and Figure 3 is a graph showing the relative activity of alginate lyase at each temperature.
The figure is a graph showing the thermal stability of alginate lyase treated at various temperatures for 20 minutes, and Figure 4 shows the alginate oligo ring passed through a column packed with the gel filtration carrier.
The elution pattern when eluted with demineralized water is shown, graph A shows the elution pattern of alginate oligo ring, and graph B shows the elution pattern of salt. Figure 1 pH Figure 2 ΔLl''C
Claims (3)
製造するに際し、アルテロモナス属に属する微生物によ
って生産される、酵素活性の至適温度が４５〜５５℃で
あるアルギン酸リアーゼを用いることを特徴とするアル
ギン酸オリゴ糖の製造方法。(1) When producing alginate oligosaccharide from sodium alginate, alginate lyase, which is produced by a microorganism belonging to the genus Alteromonas and whose optimum temperature for enzyme activity is 45 to 55°C, is used. Production method.
ナス・エスピー（Ａｌｔｅｒｏｍｏｎａｓ ｓｐ．）Ｎ
ｏ．１７８６である請求項（１）記載のアルギン酸オリ
ゴ糖の製造方法。(2) The microorganism belonging to the genus Alteromonas is Alteromonas sp.N
o. 1786. The method for producing an alginic acid oligosaccharide according to claim (1).
（分画範囲１００〜１，８００ダルトン）と接触させて
該アルギン酸オリゴ糖中に含まれる塩類等の低分子物質
を除去することを特徴とする請求項（１）又は（２）記
載の方法で製造したアルギン酸オリゴ糖の精製法。(3) The alginate oligosaccharide is brought into contact with an oligosaccharide fractionation gel filtration carrier (fraction range 100 to 1,800 daltons) to remove low molecular weight substances such as salts contained in the alginate oligosaccharide. A method for purifying alginic acid oligosaccharides produced by the method according to claim (1) or (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP02294897A JP3024994B2 (en) | 1990-10-31 | 1990-10-31 | Production method and purification method of alginate oligosaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP02294897A JP3024994B2 (en) | 1990-10-31 | 1990-10-31 | Production method and purification method of alginate oligosaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04169188A true JPH04169188A (en) | 1992-06-17 |
| JP3024994B2 JP3024994B2 (en) | 2000-03-27 |
Family
ID=17813667
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP02294897A Expired - Lifetime JP3024994B2 (en) | 1990-10-31 | 1990-10-31 | Production method and purification method of alginate oligosaccharide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3024994B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2753628A1 (en) * | 1996-09-26 | 1998-03-27 | Codif International Sa | PRODUCT FOR PREVENTING AND TREATING SKIN DISEASES, METHOD FOR PRODUCING THE SAME, AND USE THEREOF |
| WO2001040315A1 (en) * | 1999-11-30 | 2001-06-07 | Dalian Yaweite Biology Engineering Co., Ltd. | The alginate having low molecular weight, methods of manufacturing it and its use |
| EP2441458A1 (en) | 2010-08-31 | 2012-04-18 | Maruha Nichiro Foods, Inc. | A vascular protecting agent having salt-absorption inhibitory activity |
-
1990
- 1990-10-31 JP JP02294897A patent/JP3024994B2/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2753628A1 (en) * | 1996-09-26 | 1998-03-27 | Codif International Sa | PRODUCT FOR PREVENTING AND TREATING SKIN DISEASES, METHOD FOR PRODUCING THE SAME, AND USE THEREOF |
| WO1998013049A1 (en) * | 1996-09-26 | 1998-04-02 | Codif International S.A. | Method for preparing a product in particular for preventing and curing skin diseases, and resulting product |
| FR2753903A1 (en) * | 1996-09-26 | 1998-04-03 | Codif International Sa | Depolymerised sodium alginate |
| WO2001040315A1 (en) * | 1999-11-30 | 2001-06-07 | Dalian Yaweite Biology Engineering Co., Ltd. | The alginate having low molecular weight, methods of manufacturing it and its use |
| EP2441458A1 (en) | 2010-08-31 | 2012-04-18 | Maruha Nichiro Foods, Inc. | A vascular protecting agent having salt-absorption inhibitory activity |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3024994B2 (en) | 2000-03-27 |
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