JPH0561910B2 - - Google Patents
Info
- Publication number
- JPH0561910B2 JPH0561910B2 JP61068773A JP6877386A JPH0561910B2 JP H0561910 B2 JPH0561910 B2 JP H0561910B2 JP 61068773 A JP61068773 A JP 61068773A JP 6877386 A JP6877386 A JP 6877386A JP H0561910 B2 JPH0561910 B2 JP H0561910B2
- Authority
- JP
- Japan
- Prior art keywords
- endo
- type
- inulinase
- inulin
- penicillium
- 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 - Lifetime
Links
- 229920001202 Inulin Polymers 0.000 claims description 37
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 37
- 229940029339 inulin Drugs 0.000 claims description 37
- 108010090785 inulinase Proteins 0.000 claims description 34
- 241000228143 Penicillium Species 0.000 claims description 24
- 229930091371 Fructose Natural products 0.000 claims description 13
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 13
- 239000005715 Fructose Substances 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 8
- 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
- 238000012258 culturing Methods 0.000 claims description 4
- 241000233866 Fungi Species 0.000 claims description 3
- 241001558929 Sclerotium <basidiomycota> Species 0.000 claims description 3
- 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 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 241001540751 Talaromyces ruber Species 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 description 31
- 102000004190 Enzymes Human genes 0.000 description 31
- 239000000243 solution Substances 0.000 description 23
- 230000000694 effects Effects 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 238000000354 decomposition reaction Methods 0.000 description 8
- 235000000346 sugar Nutrition 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 150000002482 oligosaccharides Chemical class 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 241000282465 Canis Species 0.000 description 3
- 244000298479 Cichorium intybus Species 0.000 description 3
- 235000007542 Cichorium intybus Nutrition 0.000 description 3
- 241000464865 Penicillium trzebinskii Species 0.000 description 3
- 241001540766 Talaromyces purpureogenus Species 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101000818691 Homo sapiens Zinc finger protein 239 Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102100021121 Zinc finger protein 239 Human genes 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- 241000228195 Aspergillus ficuum Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 244000115658 Dahlia pinnata Species 0.000 description 1
- 235000012040 Dahlia pinnata Nutrition 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 240000008892 Helianthus tuberosus Species 0.000 description 1
- 235000003230 Helianthus tuberosus Nutrition 0.000 description 1
- 229910004861 K2 HPO4 Inorganic materials 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- CZMRCDWAGMRECN-UHFFFAOYSA-N Rohrzucker Natural products OCC1OC(CO)(OC2OC(CO)C(O)C(O)C2O)C(O)C1O CZMRCDWAGMRECN-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 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
- 238000002835 absorbance Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 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
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012138 yeast extract Substances 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)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、イヌリンを酵素分解してイヌロオリ
ゴ糖を効率良く生産するに適した新規エンド型イ
ヌリナーゼおよびその効率的な産生方法に関す
る。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel endo-type inulinase suitable for enzymatically degrading inulin to efficiently produce inulooligosaccharides, and to an efficient method for producing the same.
イヌリンはフラクトースが直鎖状にβ−１，２
結合で結合した、グリコース分子を末端に有する
約35個のフラクトース残基を有する多糖ポリマー
であり、種々の植物、特にキク科のダリヤ、キク
イモの塊茎、チコリーの根などに存在する。ま
た、イヌリンは熱水で容易に抽出可能であり、ビ
ート糖と同様に石灰処理などで精製できる。
Inulin is a straight chain of fructose β-1,2
It is a polysaccharide polymer having about 35 fructose residues connected by bonds and terminated with a glycose molecule, and is present in various plants, especially dahlia of the Asteraceae family, tubers of Jerusalem artichoke, and roots of chicory. Additionally, inulin can be easily extracted with hot water, and like beet sugar, it can be purified by lime treatment.
イヌリンを酸あるいは酵素で分解してフラクト
ースを得ることは以前より研究され、工業的に実
施されたこともある。酸でイヌリンを分解する場
合はPH１〜２、温度70〜90℃で数時間加水分解す
ることによりほぼ目的を達成するが、着色物の生
成やジフラクトースアンハイドライドのような副
生成物ができるなど問題がある。一方、酵素を用
いる場合はこのような問題もなく、工業的に有利
である考えられる。 Obtaining fructose by decomposing inulin with acids or enzymes has been studied for some time, and has even been carried out industrially. When decomposing inulin with acid, most of the objectives can be achieved by hydrolyzing it at a pH of 1 to 2 and a temperature of 70 to 90°C for several hours, but this can lead to the formation of colored substances and by-products such as difructose anhydride. There's a problem. On the other hand, when enzymes are used, there is no such problem and it is considered to be industrially advantageous.
ところで、イヌリンを分解する酵素（イヌリナ
ーゼ）は、イヌリン含有植物、微生物などに数多
く発見され、研究されている（文献１。後記。各
文献以下同じ）。 By the way, an enzyme that decomposes inulin (inulinase) has been discovered and studied in many inulin-containing plants, microorganisms, etc. (Reference 1, described later. The same applies to each document below).
微生物イヌリナーゼの代表的なものは、酵母で
はクルイベロミセス・フラギリス
（Kluyveromyces fragilis）やカンデイダ・ジユ
ウドトロピカリス（Candida pseudoropicalis）
のものであり、糸状菌ではアスペルギルス
（Aspergillus）属、フザリウム（Fusarium）属
やペニシリウム（Penicillium）属のものである。
これらの内でPenicillium属のエキソ型もの（文
献２）、アスペルギルス・ニガー（Asp.nigar）
のエキソ型もの（文献３）およびエンド型もの
（文献４）は精製されてその性質が調べられてい
るが、Penicillium属ではエンド型ものはいまま
では全く知られていない。 Typical microbial inulinases are yeasts such as Kluyveromyces fragilis and Candida pseudoropicalis.
Among filamentous fungi, they belong to the genus Aspergillus, Fusarium, and Penicillium.
Among these, exotypes of the genus Penicillium (Reference 2), Aspergillus niger (Asp. nigar)
Although the exo-type (Reference 3) and endo-type (Reference 4) have been purified and their properties investigated, the endo-type of Penicillium has not been known at all so far.
また、Aspergillus属カビのエンド型イヌリナ
ーゼとしてアスペルギルス・フイキユウム
（Asp.ficuum）のものが知られており、このカビ
はエキソ型のもの含んでおり、イヌリンの完全加
水分解のための両酵素の最適混合比についても調
べられている（文献５）。 In addition, Aspergillus ficuum is known as an endo-type inulinase of the Aspergillus genus mold, and this mold contains an exo-type inulinase, and an optimal mixture of both enzymes for complete hydrolysis of inulin. The ratio has also been investigated (Reference 5).
エキソ型イヌリナーゼはイヌリンのグリコース
残基の逆の側から順にフラクトースを１分子ずつ
遊離していき、一方、エンド型イヌリナーゼはイ
ヌリンの任意の位置で糖鎖を切断し、最終的にフ
ラクトース３〜６分子からなるオリゴ糖を生成す
る。また、両酵素が共存するとエンド型イヌリナ
ーゼが生成したイヌロオリゴ糖がエキソ型イヌリ
ナーゼの基質となるためフラクトースを生成する
速度が速くなることは容易に予想されるところで
あり、イヌロオリゴ糖が生成してもフラクトース
になつてしまう。 Exo-type inulinase releases fructose one molecule at a time starting from the opposite side of the glycose residue of inulin, while endo-type inulinase cleaves the sugar chain at any position on inulin, ultimately producing fructose 3-6. Produces oligosaccharides consisting of molecules. Furthermore, when both enzymes coexist, the inulooligosaccharides produced by endo-type inulinase become substrates for exo-type inulinase, so it is easy to predict that the rate of fructose production will become faster. I'm getting used to it.
イヌリナーゼの研究や利用は主としてフラクト
ースの生成を目的としてなされており、酵素を用
いてイヌリンの部分分解物、すなわちイヌロオリ
ゴ糖を積極的に得ようとする試みはなされていな
い。また、酸を用いたイヌロオリゴ糖調製の研究
も殆ど行われていない。 Research and utilization of inulinase has been carried out mainly for the purpose of producing fructose, and no attempt has been made to actively obtain a partial decomposition product of inulin, that is, inulooligosaccharide, using the enzyme. Furthermore, little research has been conducted on the preparation of inulooligosaccharides using acids.
本発明の課題は、新規なエンド型イヌリナーゼ
およびそれを得る方法を提供することである。
An object of the present invention is to provide a novel endo-type inulinase and a method for obtaining it.
本発明者等は、上記課題を解決するため、イヌ
ロオリゴ糖生産能の高い微生物を土中微生物より
検索し、意外にもPenicillium属カビにもイヌロ
オリゴ糖を生産するものがあるのを見出し、本発
明を完成した。
In order to solve the above problems, the present inventors searched soil microorganisms for microorganisms with a high ability to produce canine oligosaccharides, and unexpectedly found that some molds of the genus Penicillium also produced canine oligosaccharides. completed.
すなわち、本発明はPenicillium属カビが産生
するエンド型イヌリナーゼ、イヌリン、その部分
加水分解物、フラクトースおよびマルトースから
なる群より選ばれた少なくとも一種の化合物の存
在下に、実質的に有機質窒素の不存在下に
penicillum属カビを培養することからなるエンド
型イヌリナーゼの産生方法である。 That is, the present invention provides a method for treating bacteria in the substantial absence of organic nitrogen in the presence of at least one compound selected from the group consisting of endo-inulinase produced by Penicillium fungi, inulin, its partial hydrolyzate, fructose, and maltose. Under
This is a method for producing endo-type inulinase, which consists of culturing a fungus of the genus Penicillum.
本発明で用いるカビは、Penicillium属カビで
あり、従来はエキソ型イヌナーゼしか知られてい
なかつた。特に、本発明者らが今回検索して同定
したペニシリウム・パープロゲナム・ヴアル・ル
ブリスクレロチウム（P.purpurogenum var
rubrisclerotium）（寄託名“HOK−１”、FERM
P8705、再寄託番号：FERM BP3162）およびペ
ニシリウム・トルゼビンスキイ（P.trzebinskii）
（寄託名“HOK−２”、FERM P8706）は、以下
に述べる培養方法によるエンド型イヌリナーゼの
産生が良好であり、エキソ化型イヌリナーゼの産
生が殆どないので、得られた培養液から菌体を除
去したのみで粗酵素液として使用可能である。 The mold used in the present invention is a Penicillium genus mold, and conventionally only exo-type inunase was known. In particular, P. purpurogenum var sclerotium (P. purpurogenum var.
rubrisclerotium) (deposited name “HOK-1”, FERM
P8705, re-deposit number: FERM BP3162) and Penicillium trzebinskii (P.trzebinskii)
(Deposited name "HOK-2", FERM P8706) produces good endo-type inulinase using the culture method described below, and there is almost no production of exo-type inulinase, so bacterial cells can be isolated from the obtained culture solution. It can be used as a crude enzyme solution just by removing it.
イヌリナーゼ産生のPenicillium属カビは、天
然または人工培地として一般に使用されている各
種組成の栄養源を含む固体あるいは液体の培養基
に表面培養あるいは深層培養する。なお、イヌリ
ナーゼ産生菌の培養条件によつてはエキソ型活性
が活発となり、得られた酵素液をイヌリンに作用
させるとフラクトースばかりが得られることとな
るので、エンド型活性が主体となるように工夫す
る必要がある。その工夫としては有機質窒素をで
きるだけ用いずに培養することである。 Inulinase-producing molds of the genus Penicillium are cultured on the surface or in depth in solid or liquid culture media containing nutrient sources of various compositions, commonly used as natural or artificial media. Note that depending on the culture conditions of the inulinase-producing bacteria, the exo-type activity becomes active, and if the resulting enzyme solution is applied to inulin, only fructose will be obtained. There is a need to. The trick is to culture without using organic nitrogen as much as possible.
エキソ型とエンド型の混じつた酵素混合物から
硫安分画、種々のゲルクロマトグラフイーなどの
酵素蛋白分離技術を用いてエンド型酵素のみ分離
することは可能であるが、微生物が主としてエン
ド型酵素のみ産生することは大変好ましいことで
ある。 It is possible to separate only the endo-type enzyme from an enzyme mixture containing the exo-type and endo-type using enzyme protein separation techniques such as ammonium sulfate fractionation and various gel chromatography methods, but it is possible to isolate only the endo-type enzyme by microorganisms that mainly produce only the endo-type enzyme. It is very desirable to do so.
培養における栄養源としては、フスマ、大豆
粉、ペプトン、肉エキス、酵母エキス、コーンス
テイープリカーなどと、主たる炭素源としてフラ
クトース、イヌリンなどであり、さらにこれらを
補足する無機窒素源、リン酸塩、マグネシウム塩
および少量の金属塩を含む培地が使用される。な
お、Penicillium属カビが強力なイヌリナーゼ活
性を発現するためには有機質窒素が必要とされ、
補足する無機質窒素源としてはリン酸第１アンモ
ニウムやリン酸第２アンモニウムが効果的である
との報告がある（文献６）。 Nutrient sources for culture include bran, soybean flour, peptone, meat extract, yeast extract, corn staple liquor, etc., and main carbon sources include fructose and inulin, as well as inorganic nitrogen sources and phosphates to supplement these. , a medium containing magnesium salts and small amounts of metal salts is used. Furthermore, in order for Penicillium fungi to express strong inulinase activity, organic nitrogen is required.
It has been reported that primary ammonium phosphate and secondary ammonium phosphate are effective as supplementary inorganic nitrogen sources (Reference 6).
なお、本発明ではPenicillium属カビの成長培
養のためには他の養分と共に有機質窒素の存在が
好ましいが、培地中に産出される酵素はエンド型
活性よりもエキソ型活性が高くなり、場合によつ
てはエキソ型活性ばかりになる。そこで、上記し
たように有機質窒素をできるだけ用いずに培養す
ることが望ましく、実質的に有機質窒素を含まな
い状態でPenicillium属カビを培養すれば、初期
にはエキソ型活性を有するイヌリナーゼが産生さ
れるが、培養に伴いエンド型イヌリナーゼが主と
して産生される。なお、有機質窒素がない状態で
カビを培養する際には窒素源として各種の塩が使
用できるが、好適には上記した無機質窒素が使用
できる。 In addition, in the present invention, the presence of organic nitrogen together with other nutrients is preferable for the growth culture of Penicillium genus mold, but the enzyme produced in the medium has a higher exo-type activity than endo-type activity, and in some cases, In the end, only exo-type activity occurs. Therefore, as mentioned above, it is desirable to culture without using organic nitrogen as much as possible.If Penicillium genus mold is cultivated in a state substantially free of organic nitrogen, inulinase with exo-type activity is initially produced. However, endo-type inulinase is mainly produced during culture. Note that when culturing mold in the absence of organic nitrogen, various salts can be used as the nitrogen source, but preferably the above-mentioned inorganic nitrogen can be used.
培養温度は25〜35℃が適当であり、PHは初発は
5.0〜7.0とするが、培養中は2.5〜7.0の範囲とす
る。なお、炭素源としてイヌリンおよびその部分
加水分解物を用いた場合は培養３日目で培地のPH
は急激な低下をみるがPHの調整を行なわずに培養
する方がエンド型イヌリナーゼの活性は高くな
る。また、炭素源としてマルトースを用いた場合
のPH低下はより緩慢である。 The appropriate culture temperature is 25-35℃, and the initial pH is
5.0 to 7.0, but during culturing it should be in the range of 2.5 to 7.0. In addition, when inulin and its partial hydrolyzate are used as a carbon source, the pH of the medium changes on the third day of culture.
However, the activity of endo-type inulinase is higher when cultured without pH adjustment. Furthermore, when maltose is used as a carbon source, the pH decreases more slowly.
培養の期間は培養の条件で異なるが、エンド型
イヌリナーゼの活性が最高になる時点で培養を中
止するのが好ましく、通常４〜８日間で活性が最
高になる。 Although the culture period varies depending on the culture conditions, it is preferable to stop the culture when the endo-inulinase activity reaches its maximum, and the activity usually reaches its maximum within 4 to 8 days.
Penicillium属カビのエンド型イヌリナーゼは
菌体外に産生される酵素であるので、培養終了後
に培地から菌体を濾過あるいは遠心分離により除
去し、得られた上澄液を必要により限外濾過など
で濃縮し、硫安などによる塩析やアセトン、エタ
ノールなどの有機溶媒を加えて酵素を沈澱として
分離する。 Since the endo-inulinase of Penicillium fungi is an enzyme produced outside the bacterial cells, the bacterial cells are removed from the culture medium by filtration or centrifugation after the completion of the culture, and the resulting supernatant can be filtered by ultrafiltration if necessary. Concentrate and separate the enzyme as a precipitate by salting out with ammonium sulfate or by adding an organic solvent such as acetone or ethanol.
なお、本発明のPenicillium属カビのエンド型
イヌリナーゼは、イヌリンに対する最適分解温度
は55〜58℃にあり、従来知られている
Penicillium属カビのエキソ型イヌリナーゼのそ
れ（約45℃、文献６参照）と異なつている。 In addition, the endo-type inulinase of the Penicillium genus mold of the present invention has an optimal decomposition temperature for inulin at 55 to 58°C, which is higher than previously known.
It is different from that of exo-type inulinase of Penicillium genus mold (approximately 45°C, see Reference 6).
また、本発明のPenicillium属カビのエンド型
イヌリナーゼは熱安定性がよく、60℃／10分間の
熱安定性試験でもほとんど失活しない。 Furthermore, the endo-type inulinase of the Penicillium genus mold of the present invention has good thermal stability, and is hardly inactivated even in a thermal stability test at 60° C. for 10 minutes.
次に、本発明のPenicillium属カビのエンド型
イヌリナーゼを用いるイヌリンからイヌロオリゴ
糖を製造する方法について述べる。 Next, a method for producing inulooligosaccharide from inulin using the endo-type inulinase of Penicillium genus mold of the present invention will be described.
イヌリンの分解操作については通常イヌリンの
濃度をできるだけあげ、また、分解中の微生物汚
染を避けるために操作温度はできるだ高い方がよ
く、低くても55℃であることが望ましい。 When decomposing inulin, the concentration of inulin is usually increased as much as possible, and the operating temperature is preferably as high as possible, preferably at least 55°C, to avoid microbial contamination during decomposition.
本発明ではイヌリンの濃度を20％程度とし、反
応温度を55〜60℃、好ましくは60℃程度とし、反
応液のPHを3.5〜7.0、好ましくは4.0〜5.5として、
分解反応を行なう。 In the present invention, the concentration of inulin is about 20%, the reaction temperature is 55 to 60°C, preferably about 60°C, and the pH of the reaction solution is 3.5 to 7.0, preferably 4.0 to 5.5.
Carry out a decomposition reaction.
イヌリンの濃度は、溶液調製時の温度、原料の
イヌリンの分子量分布により変わり、広い範囲に
及びうる。特に、イヌリンを90℃以上で溶解し、
60℃に保つた場合には、25％程度の濃度でも過飽
和状態であるが調製することができる。 The concentration of inulin varies depending on the temperature during solution preparation and the molecular weight distribution of the raw material inulin, and can range over a wide range. In particular, inulin is dissolved at 90℃ or higher,
When kept at 60°C, it is possible to prepare a supersaturated state even at a concentration of about 25%.
本発明により、Penicillium属カビのエンド型
イヌリナーゼが提供され、イヌリンから効果的に
イヌロオリゴ糖をフラクトースの生成なしに製造
することがてきるようになり、工業的に極めて優
れたイヌロオリゴ糖の製造方法が提供される。 According to the present invention, endo-type inulinase from Penicillium genus mold is provided, and it has become possible to effectively produce inulooligosaccharides from inulin without producing fructose, and an industrially excellent method for producing inulooligosaccharides has been established. provided.
以下、実施例により本発明を説明する。 The present invention will be explained below with reference to Examples.
イヌリン分解酵素の力価測定法
1.5％イヌリン溶液0.5mlに１／10M酢酸緩衝液
（PH5.0）で希釈した酵素液0.5mlを加え、50℃で
30分間静置後、生成した還元糖を３，５−ジニト
ロサリチル酸法により500nmの吸光度で比色定量
した。酵素単位は50℃において酵素液１mlあたり
１分間に1μmoleの還元糖を生成する酵素量を
1unitとした。 Inulin-degrading enzyme titer measurement method Add 0.5 ml of enzyme solution diluted with 1/10M acetate buffer (PH5.0) to 0.5 ml of 1.5% inulin solution and incubate at 50℃.
After standing for 30 minutes, the resulting reducing sugar was colorimetrically determined using the 3,5-dinitrosalicylic acid method using absorbance at 500 nm. The enzyme unit is the amount of enzyme that produces 1 μmole of reducing sugar per minute per ml of enzyme solution at 50℃.
It was set as 1 unit.
実施例 １
P.purpurogenum var rubrisclerotium（寄託名
“HOK−１”、FERM P8705、再寄託番号：
FERM BP3162によるエンド型イヌリナーゼ
の調製
イヌリン（チコリー根より分離精製したもの）
0.5％、マルトース0.5％、NH4H2PO40.27％、K2
HPO40.1％、MgSO4・7H2O0.05％、
Tween800.02％の培地を水道水で調製し、PHを７
に調整したのち、500ml容の坂口フラスコに100ml
とり、滅菌処理した後菌体を１白金耳接種し、28
℃で96時間振盪培養した。培養液から漏過により
菌体を除去して、イヌリンの分解活性を測定した
ところ2.5unit／ml培養液であつた。Example 1 P. purpurogenum var rubrisclerotium (deposit name “HOK-1”, FERM P8705, re-deposit number:
Preparation of endo-type inulinase using FERM BP3162 Inulin (separated and purified from chicory root)
0.5% , Maltose 0.5%, NH4H2PO4 0.27 %, K2
HPO4 0.1%, MgSO4・7H2O0.05 %,
Prepare a Tween 800.02% medium with tap water and adjust the pH to 7.
After adjusting to 100ml into a 500ml Sakaguchi flask.
After sterilization, one platinum loopful of bacterial cells was inoculated, and 28
The cells were cultured with shaking at ℃ for 96 hours. Bacterial cells were removed from the culture solution by leakage, and the inulin degrading activity was measured, and it was found to be 2.5 units/ml of the culture solution.
上記して得られた菌体を除去した培養液1000ml
（イヌリンの分解活性2.5unit／ml）に硫安を添加
し、40〜80％飽和沈澱画分を遠心分離し、１／
10M酢酸緩衝液（PH5.0）に溶解し、蒸留水に対
して透析して、イヌリンの分解活性17.7unit／ml
の酵素液100ml得た。 1000ml of the culture solution obtained above from which the bacterial cells have been removed
(inulin degrading activity 2.5 units/ml) was added with ammonium sulfate, and the 40-80% saturated precipitate fraction was centrifuged.
Dissolved in 10M acetate buffer (PH5.0) and dialyzed against distilled water, the degrading activity of inulin was 17.7 units/ml.
100ml of enzyme solution was obtained.
得られた酵素は、SDSポリアクリルアミドゲル
電気泳動法で測定した分子量55000±3000、酵素
活性の至適PHおよび温度4.5〜5.5、約55℃であ
り、60℃／10分の熱安定試験でも活性の低下は認
められない。 The obtained enzyme has a molecular weight of 55,000 ± 3,000 as measured by SDS polyacrylamide gel electrophoresis, an optimum pH and temperature for enzyme activity of 4.5 to 5.5, approximately 55°C, and is also active in a thermal stability test at 60°C for 10 minutes. No decrease was observed.
参考例 １
イヌロオリゴ糖の製造 (1)
実施例１で得られた酵素液を20％イヌリン溶液
に、イヌリン１ｇあたり3unit添加し、PH4.5、60
℃で48時間反応した。Reference Example 1 Production of inulooligosaccharide (1) Add 3 units of the enzyme solution obtained in Example 1 to a 20% inulin solution per 1 g of inulin, and adjust the pH to 4.5 and 60.
The reaction was carried out at ℃ for 48 hours.
反応の途中で反応液を取り、ゲルクロマトグラ
フイで分析したところ、反応開始３時間後で重合
度（DP）３〜９のイヌロオリゴ糖と少量のDP２
およびDP１の糖が生成していることがわかり
（第１図）、24時間ではDP３〜９のイヌロオリゴ
糖の生成量が増していた（第２図）。反応停止時
（48時間反応後）では、DP１が1.5％、DP２が3.3
％、DP３が31.4％、DP４が26.6％、DP５が20.4
％、DP６が13.3％、それ以上のもの3.5％であつ
た（第３図）。 When the reaction solution was taken during the reaction and analyzed by gel chromatography, it was found that 3 hours after the start of the reaction, inulooligosaccharides with a degree of polymerization (DP) of 3 to 9 and a small amount of DP2 were detected.
It was found that sugars of DP1 and DP1 were produced (Fig. 1), and the amount of canine oligosaccharides of DP3 to 9 was increased after 24 hours (Fig. 2). When the reaction stopped (after 48 hours of reaction), DP1 was 1.5% and DP2 was 3.3%.
%, DP3 31.4%, DP4 26.6%, DP5 20.4
%, DP6 was 13.3%, and DP6 was 3.5% (Figure 3).
このような経時的分解パターンは典型的なエン
ド型酵素の分解パターンであり、この酵素液によ
る最終的な分解限度は果糖として計算して約50％
であつた。 This decomposition pattern over time is a typical decomposition pattern of endo-type enzymes, and the final decomposition limit by this enzyme solution is approximately 50% when calculated as fructose.
It was hot.
反応終了後、イオン交換樹脂や活性炭を用いて
常法により生成したのち、濃縮乾燥して白色のイ
ヌロオリゴ糖を得ることができた。 After the reaction was completed, it was produced using an ion exchange resin or activated carbon in a conventional manner, and then concentrated and dried to obtain a white inulooligosaccharide.
実施例 ２
P.trzebinskii（寄託名“HOK−２”、FERM
P8706）によるエンド型イヌリナーゼの調製
イヌリン（チコリー根より分離生成したもの）
1.0％、（NH4）2HPO40.15％、KH2PO40.1％、
Tween800.02％、MgSO4・7H2O0.05％を含む水
道水で調製したPH７の培地を１容の三角フラス
コに300mlとり、滅菌処理した後、菌体を１白金
耳接種し、28℃で振幅７cm、ストローク数100回
転培養した。Example 2 P.trzebinskii (deposited name “HOK-2”, FERM
Preparation of endo-type inulinase using P8706) Inulin (isolated from chicory root)
1.0%, ( NH4 ) 2HPO4 0.15 %, KH2PO4 0.1 %,
Add 300 ml of a PH7 medium prepared with tap water containing 0.02% Tween 800.02% and 0.05% MgSO 4 7H 2 O to a 1-volume Erlenmeyer flask, sterilize it, then inoculate one loopful of bacterial cells at 28°C. Culture was performed with an amplitude of 7 cm and a number of strokes of 100 revolutions.
培養４日目以降にPHが低下をはじめ、６日目で
はPHが3.5となつた。始めはフラクトース生成型
（エキソ型）であつたイヌリナーゼ活性はイヌロ
オリゴ糖生成型（エンド型）となつた。７日目に
培養液１mlあたり0.8unitと最大になつた時点で
培養を止め、培養液と菌体を漏別した。 After the 4th day of culture, the PH began to decrease and reached 3.5 on the 6th day. Inulinase activity, which was initially fructose-producing (exo-type), changed to inulooligosaccharide-producing (endo-type). On the 7th day, when the culture reached a maximum of 0.8 units per ml of culture solution, the culture was stopped, and the culture solution and bacterial cells were separated.
実施例１と同様に培養液を塩析し、硫安による
40〜80％飽和沈澱画分を得、１／100N酢酸緩衝
液（PH5.0）に溶解した後、バイオラツド社製の
バイオゲルＰ−6DGを用いて脱塩し、粗酵素液
を得た。 The culture solution was salted out in the same manner as in Example 1, and salted out with ammonium sulfate.
A 40-80% saturated precipitate fraction was obtained, dissolved in 1/100N acetate buffer (PH5.0), and then desalted using Biogel P-6DG manufactured by BioRad to obtain a crude enzyme solution.
参考例 ２
イヌロオリゴ糖の製造 (2)
実施例２で得られた酵素液20％イヌリン溶液
に、イヌリン１ｇあたり3unit添加し、PH4.5、60
℃で48時間反応した。Reference Example 2 Production of inulooligosaccharide (2) 3 units per 1 g of inulin were added to the 20% inulin solution of the enzyme solution obtained in Example 2, and PH4.5, 60
The reaction was carried out at ℃ for 48 hours.
反応終了後、ゲルクロマトグラフイで分析した
ところ、DP１が1.3％、DP２が0.9％、DP３が
26.5％、DP４が27.6％、DP５が18.5％、DP６が
14.2％、それ以上のもの11.0％であつた（第４
図）。 After the reaction was completed, gel chromatography analysis revealed that DP1 was 1.3%, DP2 was 0.9%, and DP3 was 0.9%.
26.5%, DP4 27.6%, DP5 18.5%, DP6
14.2%, and 11.0% higher (4th
figure).
本菌株の粗酵素液によりイヌリンを分解した場
合の経時的な分解パターンは、実施例１と同様に
エンド型の分解パターンを示した。 When inulin was degraded using the crude enzyme solution of this strain, the degradation pattern over time showed an endo-type degradation pattern as in Example 1.
文献１： Vandemme，E.J.，Derycke，D.G.；
Adv.Appl.Microbiol.，1983，29，P139〜
176.
文献２： 中村豊彦、中津誠一郎；農化、1977，
51，P681〜689.
文献３： 中村豊彦ら；農化、1978，52，P159
〜166.
文献４： 中村豊彦ら；農化、1978，52，P581
〜587.
文献５： J.Zitten；Starch、1981，33，P373〜
377.
文献６： 中村豊彦ら；農化、1969，43，P599
〜605.Reference 1: Vandemme, EJ, Derycke, DG;
Adv.Appl.Microbiol., 1983, 29, P139~
176. Reference 2: Toyohiko Nakamura, Seiichiro Nakatsu; Noka, 1977,
51, P681-689. Reference 3: Toyohiko Nakamura et al.; Noka, 1978, 52, P159
~166. Reference 4: Toyohiko Nakamura et al.; Noka, 1978, 52, P581
~587. Reference 5: J. Zitten; Starch, 1981, 33, P373~
377. Reference 6: Toyohiko Nakamura et al.; Noka, 1969, 43, P599
~605.
第１図〜第４図はいずれも本発明の酵素により
イヌリンを加水分解したときの糖成分のゲルクロ
マトグラフである。第１図〜第３図は参考例１の
経時分析結果を示し（第１図反応開始３時間後、
第２図同24時間後、第３図同48時間後）、第４図
は参考例２の酵素分解終了後の分析結果である。
なお、基線のしたの数字は糖の重合度（DP）
を表す。
Figures 1 to 4 are gel chromatographs of sugar components when inulin is hydrolyzed by the enzyme of the present invention. Figures 1 to 3 show the results of time-lapse analysis of Reference Example 1 (Figure 1: 3 hours after the start of the reaction,
Figure 2 (24 hours later, Figure 3 48 hours later) and Figure 4 show the analysis results of Reference Example 2 after the enzymatic decomposition was completed. The number below the baseline is the degree of polymerization (DP) of the sugar.
represents.
Claims (1)
するエンド型イヌリナーゼ。 ２ Penicillium属カビがペニシリウム・パープ
ロゲナム・ヴアル・ルブリスクレロチウム（P.
purpurogenum var rubrisclerotium）である特
許請求の範囲第１項記載のエンド型イヌリナー
ゼ。 ３ Penicillium属カビがペニシリウム・トルゼ
ビンスキイ（P.truzebinskii）である特許請求の
範囲第１項記載のエンド型イヌリナーゼ。 ４ イヌリン、その部分加水分解物、フラクトー
スおよびマルトースからなる群より選ばれた少な
くとも一種の化合物の存在下に、実質的に有機質
窒素の不存在下に、Penicillium属カビを培養す
ることからなるエンド型イヌリナーゼの産生方
法。 ５ Penicillium属カビがペニシリウム・パープ
ロゲナム・ヴアル・ルブリスクレロチウム（P.
purpurogenum var rubrisclerotium）である特
許請求の範囲第４項記載のエンド型イヌリナーゼ
の産生方法。 ６ Penicillium属カビがペニシリウム・トリゼ
ビンスキイ（P.truzebinskii）である特許請求の
範囲第４項記載のエンド型イヌリナーゼの産生方
法。[Scope of Claims] 1. Endo-type inulinase produced by a fungus of the genus Penicillium. 2 The Penicillium genus mold is Penicillium perprogenum val rubris sclerotium (P.
The endo-type inulinase according to claim 1, which is P. purpurogenum var rubrisclerotium. 3. The endo-type inulinase according to claim 1, wherein the Penicillium genus mold is P. truzebinskii. 4 Endo-type, which consists of culturing Penicillium genus mold in the presence of at least one compound selected from the group consisting of inulin, its partial hydrolyzate, fructose, and maltose, and in the substantial absence of organic nitrogen. Method for producing inulinase. 5 The Penicillium genus mold is Penicillium perprogenum val rubris sclerotium (P.
5. The method for producing endo-type inulinase according to claim 4, which is P. purpurogenum var rubrisclerotium. 6. The method for producing endo-type inulinase according to claim 4, wherein the Penicillium genus mold is P. truzebinskii.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61068773A JPS62228293A (en) | 1986-03-28 | 1986-03-28 | Production of inulooligosaccharide |
| JP5021074A JPH06102033B2 (en) | 1986-03-28 | 1993-02-09 | Method for producing inulooligosaccharide |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61068773A JPS62228293A (en) | 1986-03-28 | 1986-03-28 | Production of inulooligosaccharide |
| JP5021074A JPH06102033B2 (en) | 1986-03-28 | 1993-02-09 | Method for producing inulooligosaccharide |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5021074A Division JPH06102033B2 (en) | 1986-03-28 | 1993-02-09 | Method for producing inulooligosaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62228293A JPS62228293A (en) | 1987-10-07 |
| JPH0561910B2 true JPH0561910B2 (en) | 1993-09-07 |
Family
ID=26358090
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61068773A Granted JPS62228293A (en) | 1986-03-28 | 1986-03-28 | Production of inulooligosaccharide |
| JP5021074A Expired - Lifetime JPH06102033B2 (en) | 1986-03-28 | 1993-02-09 | Method for producing inulooligosaccharide |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5021074A Expired - Lifetime JPH06102033B2 (en) | 1986-03-28 | 1993-02-09 | Method for producing inulooligosaccharide |
Country Status (1)
| Country | Link |
|---|---|
| JP (2) | JPS62228293A (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE1010449A3 (en) * | 1996-08-01 | 1998-08-04 | Raffinerie Tirlemontoise Sa | PROCESS FOR PREPARING A COMPOSITION SACCHARIDES POLYDIPERSEE, COMPOSITION polydispersed saccharide, PRODUCTS, PHARMACEUTICAL AND / OR COSMETIC COMPOSITION COMPRISING polydisperse. |
| DE19717823A1 (en) * | 1997-04-26 | 1998-10-29 | Tshisuaka Barbara I Dr | A process for producing an endoinulinase-producing microorganism, a process for producing endoinulinase and a process for producing oligofructosides from insulin and a process for detecting endoinulinase activity |
| CN112980813B (en) * | 2021-01-13 | 2022-08-30 | 云南师范大学 | Low-temperature modified exoinulase mutant MutS117G |
| CN112813053B (en) * | 2021-01-13 | 2022-06-24 | 云南师范大学 | Inulase mutant MutY119H and preparation method thereof |
| CN112725306B (en) * | 2021-01-13 | 2022-06-24 | 云南师范大学 | Inulase mutant MutY119T with changed thermal salinity and application thereof |
| CN112813052B (en) * | 2021-01-13 | 2022-08-26 | 云南师范大学 | Exo-inulase mutant MutDP121ET6 with improved low-temperature activity |
| CN112813050B (en) * | 2021-01-13 | 2022-08-30 | 云南师范大学 | Exo-inulinase mutant MutP126Q with reduced thermostability |
| CN112725307B (en) * | 2021-01-13 | 2022-09-16 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutG169 delta 4 with reduced heat resistance and application thereof |
-
1986
- 1986-03-28 JP JP61068773A patent/JPS62228293A/en active Granted
-
1993
- 1993-02-09 JP JP5021074A patent/JPH06102033B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0614792A (en) | 1994-01-25 |
| JPH06102033B2 (en) | 1994-12-14 |
| JPS62228293A (en) | 1987-10-07 |
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