JPS6328389A - Beta-glucosidase and production thereof - Google Patents
Beta-glucosidase and production thereofInfo
- Publication number
- JPS6328389A JPS6328389A JP17086786A JP17086786A JPS6328389A JP S6328389 A JPS6328389 A JP S6328389A JP 17086786 A JP17086786 A JP 17086786A JP 17086786 A JP17086786 A JP 17086786A JP S6328389 A JPS6328389 A JP S6328389A
- Authority
- JP
- Japan
- Prior art keywords
- glucose
- glucosidase
- enzyme
- beta
- streptomyces
- 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.)
- Pending
Links
- 102000006995 beta-Glucosidase Human genes 0.000 title claims abstract description 22
- 108010047754 beta-Glucosidase Proteins 0.000 title claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 37
- 239000008103 glucose Substances 0.000 claims abstract description 37
- 230000000694 effects Effects 0.000 claims abstract description 19
- 241000187747 Streptomyces Species 0.000 claims abstract description 8
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 239000013587 production medium Substances 0.000 claims description 2
- 102000004366 Glucosidases Human genes 0.000 claims 1
- 108010056771 Glucosidases Proteins 0.000 claims 1
- 241000187180 Streptomyces sp. Species 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract description 3
- NEZJDVYDSZTRFS-RMPHRYRLSA-N Phenyl beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1 NEZJDVYDSZTRFS-RMPHRYRLSA-N 0.000 abstract description 2
- 238000005273 aeration Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 239000012531 culture fluid Substances 0.000 abstract 2
- 230000000813 microbial effect Effects 0.000 abstract 2
- 150000001720 carbohydrates Chemical class 0.000 abstract 1
- 238000012834 spinner culture method Methods 0.000 abstract 1
- 239000012134 supernatant fraction Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 53
- 108090000790 Enzymes Proteins 0.000 description 53
- 229940088598 enzyme Drugs 0.000 description 53
- 241000894006 Bacteria Species 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 108010059892 Cellulase Proteins 0.000 description 9
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose 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)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 9
- 229940106157 cellulase Drugs 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 244000144725 Amygdalus communis Species 0.000 description 6
- 235000020224 almond Nutrition 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000001913 cellulose Substances 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 238000000691 measurement method Methods 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- HIWPGCMGAMJNRG-ACCAVRKYSA-N Sophorose Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HIWPGCMGAMJNRG-ACCAVRKYSA-N 0.000 description 4
- 239000008351 acetate buffer Substances 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- HIWPGCMGAMJNRG-UHFFFAOYSA-N beta-sophorose Natural products OC1C(O)C(CO)OC(O)C1OC1C(O)C(O)C(O)C(CO)O1 HIWPGCMGAMJNRG-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- PZDOWFGHCNHPQD-VNNZMYODSA-N sophorose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O PZDOWFGHCNHPQD-VNNZMYODSA-N 0.000 description 4
- IFBHRQDFSNCLOZ-RMPHRYRLSA-N 4-nitrophenyl beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-RMPHRYRLSA-N 0.000 description 3
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 3
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 3
- 239000007994 TES buffer Substances 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- -1 methyl-β-glucoside Chemical compound 0.000 description 3
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 3
- 229940120668 salicin Drugs 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 2
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 description 2
- 235000011437 Amygdalus communis Nutrition 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000007987 MES buffer Substances 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 2
- 229940093496 esculin Drugs 0.000 description 2
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000295 fuel oil Substances 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- RPWPRIVEZXDLST-RMPHRYRLSA-N (2r,3r,4s,5s,6r)-6-(hydroxymethyl)-2-(4-nitrophenyl)oxane-2,3,4,5-tetrol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@]1(O)C1=CC=C([N+]([O-])=O)C=C1 RPWPRIVEZXDLST-RMPHRYRLSA-N 0.000 description 1
- QIGJYVCQYDKYDW-UHFFFAOYSA-N 3-O-alpha-D-mannopyranosyl-D-mannopyranose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(CO)OC(O)C1O QIGJYVCQYDKYDW-UHFFFAOYSA-N 0.000 description 1
- 241001156739 Actinobacteria <phylum> Species 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose 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](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- AYRXSINWFIIFAE-UHFFFAOYSA-N O6-alpha-D-Galactopyranosyl-D-galactose Natural products OCC1OC(OCC(O)C(O)C(O)C(O)C=O)C(O)C(O)C1O AYRXSINWFIIFAE-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960000271 arbutin Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- DLRVVLDZNNYCBX-CQUJWQHSSA-N gentiobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-CQUJWQHSSA-N 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- QIGJYVCQYDKYDW-LCOYTZNXSA-N laminarabiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1O QIGJYVCQYDKYDW-LCOYTZNXSA-N 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 150000002897 organic nitrogen compounds Chemical class 0.000 description 1
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 150000008495 β-glucosides Chemical class 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
近年、セルロースが安価なグルコース源として脚光をあ
び、これを発酵原料として工業用アルコールなどを大量
に安定供給する技術の開発が進められている。本発明は
このセルロースを効率よく分解しうる酵素を提供するも
のである。[Detailed Description of the Invention] [Industrial Application Field] In recent years, cellulose has been in the spotlight as an inexpensive source of glucose, and technology is being developed to stably supply large amounts of industrial alcohol etc. using cellulose as a fermentation raw material. . The present invention provides an enzyme that can efficiently decompose cellulose.
セルロースからグルコースを製造する方法のひとつにセ
ルラーゼを用いた酵素法がある。このセルラーゼには反
応を効率よく行なわせるために一般に複合酵素系が利用
され、セルラーゼ反応の中間生成物であるセロビオース
からグルコースへの反応はβ−グルコシダーゼが分担し
ている。One of the methods for producing glucose from cellulose is an enzymatic method using cellulase. A complex enzyme system is generally used for this cellulase in order to carry out the reaction efficiently, and β-glucosidase is responsible for the reaction from cellobiose, which is an intermediate product of the cellulase reaction, to glucose.
しかしながら、従来のセルラーゼでは多量の酵素を用い
ても実用レベルの高濃度のグルコースを生成させるため
の条件ではセルロースを100%糖化することはできず
、特に反応の後半においてグルコースの生成速度が大幅
に低下するという問題があった。本発明者らはこのよう
な問題点を解決するべく鋭意検討の結果、セルラーゼ複
合酵素系のうち最終段階の反応を受は持つβ−グルコシ
ダーゼが生成物であるグルコースによって強く阻害を受
けることを動力学的解析により確認した。However, with conventional cellulase, even if a large amount of enzyme is used, it is not possible to saccharify cellulose 100% under the conditions required to generate glucose at a high concentration at a practical level, and the rate of glucose production is significantly reduced, especially in the latter half of the reaction. There was a problem with the decline. As a result of intensive studies to solve these problems, the present inventors discovered that β-glucosidase, which is responsible for the final stage of the reaction in the cellulase complex enzyme system, is strongly inhibited by the product glucose. This was confirmed by scientific analysis.
そこで、グルコース阻害の少ないβ−グルコシダーゼを
取得するべ〈従来のセルラーゼ産生菌の変異操作や細胞
融合を行なったが満足しうる結果は得られなかった。Therefore, in order to obtain β-glucosidase with less glucose inhibition, conventional mutagenesis and cell fusion of cellulase-producing bacteria were performed, but no satisfactory results were obtained.
本発明者らはこのような問題点を解決するべくさらに検
討を進め、新たに自然界から分離したストレプトミセス
属に属する微生物がこの目的とする酵素を産生しうろこ
とを見出し、この知見に基いて本発明を完成するに至っ
た。すなわち、本発明は、1Mグルコース存在下の相対
活性がグルコース不在時の65%以上であるβ−グルコ
シダーゼとこの酵素を産生しうるストレプトミセス属微
生物を用いてこの酵素を製造する方法に関するものであ
る。In order to solve these problems, the present inventors conducted further studies and discovered that a microorganism belonging to the genus Streptomyces, which was newly isolated from nature, can produce the desired enzyme, and based on this knowledge, The present invention has now been completed. That is, the present invention relates to a β-glucosidase whose relative activity in the presence of 1M glucose is 65% or more of that in the absence of glucose, and a method for producing this enzyme using a Streptomyces microorganism capable of producing this enzyme. .
次に、実施例で得られた本酵素の理化学的性質を示す。Next, the physicochemical properties of the present enzyme obtained in Examples will be shown.
(1)作用及び基質特異性
本酵素の各種基質に対する作用を測定した結果を下表に
示す。(1) Action and substrate specificity The results of measuring the action of this enzyme on various substrates are shown in the table below.
測定方法は、表に示した濃度の各基質、20μg蛋白の
AJ 9456又は25μg蛋白のAJ 9457の酵
素、及び0.1 M MES (2(N −Morph
olino) ethane−sulfonic ac
id) pH6,5緩衝液を含む全容積0.2)111
の反応液を50℃で1時間反応させ、生成したグルコー
スをグルコースオキシダーゼを用いた比色定量法で測定
することによって求めた。表中の数字はセロビオースに
ついて得られた値を100とした相対値で表示した。従
来品にはアーモンド由来のβ−グルコシダーゼを用いた
。表から明らかなように本酵素は従来品と比較するとソ
ホロースとサリシンに対する作用が太き(異なっている
。The measurement method consisted of each substrate at the concentrations shown in the table, 20 μg protein of AJ 9456 or 25 μg protein of AJ 9457 enzyme, and 0.1 M MES (2(N-Morph)).
olino) ethane-sulfonic ac
id) Total volume containing pH 6,5 buffer 0.2) 111
The reaction solution was reacted at 50° C. for 1 hour, and the glucose produced was determined by colorimetric determination using glucose oxidase. The numbers in the table are expressed as relative values with the value obtained for cellobiose taken as 100. The conventional product used β-glucosidase derived from almonds. As is clear from the table, this enzyme has stronger (different) effects on sophorose and salicin than conventional products.
すなわち、ソホロースに対する活性は従来品の2以下で
あり、サリシンに対する活性は1/10以下である。That is, the activity against sophorose is 2 or less of the conventional product, and the activity against salicin is 1/10 or less.
(2) 至適pH
AJ 9456菌から得られた酵素の至適pH曲線を第
1図にそしてAJ 9457菌から得られた至適pH曲
線を第2図に示す。測定方法としては、基質に5mMp
−ニトロフェニル−β−グルコシドヲ用い、各pHにお
いて50℃で10分間反応後生成したp−ニトロフェノ
ール量を400nmにおける吸光度を測定して求めた。(2) Optimal pH The optimum pH curve of the enzyme obtained from AJ 9456 bacteria is shown in FIG. 1, and the optimum pH curve obtained from AJ 9457 bacteria is shown in FIG. The measurement method is to add 5mMp to the substrate.
Using -nitrophenyl-β-glucoside, the amount of p-nitrophenol produced after reaction at 50° C. for 10 minutes at each pH was determined by measuring the absorbance at 400 nm.
図中丸印は0.1 Mクエン酸−リン酸緩衝液を、×印
は0.1MMES緩衝液を、そして三角印は0.1 M
TES (N −Tris (Hydroxy−me
thyl) nethyl −2−aminoetha
nesulfonic acid)緩衝液を用いた場合
をそれぞれ示している。これらの図に示すように本酵素
の至適pHはいずれもpH6〜6.5にある。尚、同じ
条件で測定したアーモンド由来のβ−グルコシダーゼの
至適pHは5.5であった。In the figure, the circles indicate 0.1 M citrate-phosphate buffer, the x marks indicate 0.1 MMES buffer, and the triangle marks indicate 0.1 M
TES (N-Tris (Hydroxy-me)
thyl) nethyl-2-aminoetha
nesulfonic acid) buffer is used. As shown in these figures, the optimum pH of this enzyme is between pH 6 and 6.5. The optimum pH of almond-derived β-glucosidase measured under the same conditions was 5.5.
(3)安定pH範囲
AJ 9456菌から得られた酵素のpH安定性を示す
曲線を第3図に丸印でそしてAJ 9457菌から得ら
れた酵素のpH安定性を示す曲線を同図に×印でそれぞ
れ示す。測定方法としては、各pHにおいて50°Cで
20分間加温し、残存活性を測定することにより求めた
。用いた緩衝液はpH3〜6は0.1Mクエン酸−リン
酸緩衝液、pH7〜8は0、1 M TES緩衝液、p
H9〜10は0.1 M トリス緩衝液、そしてpH1
1は0.1Mグリシン−NaOH緩衝液である。図に示
すように本酵素の安定pH範囲はいずれもpH5〜9.
5にある。(3) Stable pH range The curve showing the pH stability of the enzyme obtained from the AJ 9456 bacterium is marked with a circle in Figure 3, and the curve showing the pH stability of the enzyme obtained from the AJ 9457 bacterium is marked with an x in the same figure. Each is indicated by a mark. The measurement method was to heat each pH at 50°C for 20 minutes and measure the residual activity. The buffers used were 0.1 M citrate-phosphate buffer for pH 3 to 6, 0 and 1 M TES buffer for pH 7 to 8, and p
H9-10 is 0.1 M Tris buffer and pH 1
1 is a 0.1M glycine-NaOH buffer. As shown in the figure, the stable pH range of this enzyme is pH 5-9.
It is in 5.
(4)力価の測定法
0.1M酢酸緩衝液、pH5,0,5mMp−ニトロフ
ェニル−β−グルコサイドと酵素を含む全容1mlを6
0℃10分間反応させて1 mlの1MN82CO1を
加えて反応を停止し、生成したp−ニトロフェノール量
を400nmでの吸光度の測定により求めた。この条件
で1分間当り1μmol のp−ニトロフェノールを生
成させる酵素量をl unitとした。(4) Measurement method of titer: 1 ml of 0.1 M acetate buffer, pH 5, containing 0.5 mM p-nitrophenyl-β-glucoside and enzyme.
The reaction was allowed to proceed for 10 minutes at 0°C, and 1 ml of 1M N82CO1 was added to stop the reaction, and the amount of p-nitrophenol produced was determined by measuring absorbance at 400 nm. The amount of enzyme that produced 1 μmol of p-nitrophenol per minute under these conditions was defined as l unit.
(5)熱安定性
p H5,0の0.1M酢酸緩衝液中で各温度で1時間
加熱後の残存活性を測定した結果を第4図に示す。図中
、白丸はAJ 9456菌から得られた酵素を、X印は
AJ 9457菌から得られた酵素を、そして黒丸はア
ーモンド由来の酵素をそれぞれ表わしている。この図に
示すようにAJ 9456菌から得られた酵素は60°
c、 AJ9457菌から得られた酵素は55°Cま
で安定である。それに対し、アーモンド由来の酵素の熱
安定性は50℃までであった。(5) Thermal stability The results of measuring the residual activity after heating at each temperature for 1 hour in a 0.1M acetate buffer at pH 5.0 are shown in FIG. In the figure, white circles represent enzymes obtained from AJ 9456 bacteria, X marks represent enzymes obtained from AJ 9457 bacteria, and black circles represent enzymes derived from almonds. As shown in this figure, the enzyme obtained from AJ 9456 bacterium is 60°
c. The enzyme obtained from AJ9457 bacteria is stable up to 55°C. In contrast, the thermostability of the almond-derived enzyme was up to 50°C.
(6) グルコース阻害定数
本酵素及び従来のβ−グルコシダーゼについてグルコー
ス阻害定数を測定した結果を下表に示す。(6) Glucose inhibition constant The results of measuring the glucose inhibition constant for the present enzyme and conventional β-glucosidase are shown in the table below.
反応はpH5,0の0.1M酢酸緩衝液中で行なわせs
Lineweaver−Burkプロットを利用して
阻害定数を求めた。The reaction was carried out in 0.1M acetate buffer at pH 5.0.
Inhibition constants were determined using Lineweaver-Burk plot.
(7) グルコースン農度の影古
本酵素の活性に対するグルコースの影響を測定した結果
を第5図に示す。図中AはAJ 9456菌の洗浄菌体
そのものを酵素として使用した場合を示し、Bは超音波
で菌体を破壊して抽出した菌細胞抽出液を示す。Cはア
ーモンド由来の酵素をそしてDは従来優秀な酵素である
とされているスボロトリクムセルロフィルム由来の酵素
を示している。(7) Effect of Glucose Acidity Figure 5 shows the results of measuring the influence of glucose on the activity of the enzyme. In the figure, A shows the case where the washed cells of the AJ 9456 bacterium itself were used as the enzyme, and B shows the bacterial cell extract obtained by destroying the cells with ultrasound. C indicates an enzyme derived from almonds, and D indicates an enzyme derived from Suborotrichum cellulophilum, which has been considered to be an excellent enzyme.
測定方法は0.1M酢酸緩衝液、p H5,0,5mM
p−ニトロフェニル−β−グルコサイドと酵素を含む全
容積1 mj!を60℃10分閘反応させて上記に示し
た方法で400nmでの吸光度を測定した。Measurement method: 0.1M acetate buffer, pH 5, 0.5mM
Total volume containing p-nitrophenyl-β-glucoside and enzyme 1 mj! was reacted at 60° C. for 10 minutes, and the absorbance at 400 nm was measured using the method described above.
同図に示すように本酵素は1Mグルコース存在下におい
ても初期の65%以上、特に80%以上の活性を保持し
ているがアーモンド由来のものは約60%にそしてスボ
ロトリクム由来の酵素に至っては10%以下に低下して
いる。As shown in the figure, this enzyme retains more than 65% of its initial activity even in the presence of 1M glucose, especially more than 80% of its initial activity, but the activity of the enzyme derived from almonds is about 60%, and that of the enzyme derived from Suborotrichum. It has decreased to below 10%.
以上の如く、本酵素は従来のβ−グルコシダーゼと理化
学的性質が大きく異なり、持にグルコースによる活性低
化が少ないことで従来の酵素と明らかに異なる。そこで
本酵素を新規酵素であると認定するに至った。As described above, this enzyme has significantly different physicochemical properties from conventional β-glucosidase, and is clearly different from conventional enzymes in that its activity is less likely to be lowered by glucose. Therefore, this enzyme was recognized as a new enzyme.
かかる本酵素は例えばストレプトミセス・エスピーAJ
9456 (FER?I P−8861)、同^J
9457などを培養することにより産生させることがで
きる。Such this enzyme is, for example, Streptomyces sp.
9456 (FER?I P-8861), same^J
It can be produced by culturing 9457 and the like.
ストレプトミセス・エスピーAJ 9456及び同AJ
9457の菌学的性質を次に示す。Streptomyces sp AJ 9456 and AJ
The mycological properties of 9457 are shown below.
1形態的性質
l5P−1寒天平板培地、50℃、7日間培養したもの
の形態的性質
基中菌糸および気菌糸を有する。1. Morphological properties: Cultured on 15P-1 agar plate medium at 50°C for 7 days. Morphological properties: Medium hyphae and aerial hyphae.
気菌糸上の胞子柄に分節胞子の連鎖を形成する。Forms chains of segmented spores on sporophores on aerial hyphae.
胞子柄はらせん状ないし直線状を呈する。The sporophyte is spiral or straight.
2細胞壁のタイプ
l5P−II寒天平板培地、50゛C17日間培養した
菌体を集め、6Nl(Cβにて100℃、18時間加水
分解し、濾過により残渣を除去した上滑液を「放線菌の
同定実験法」 (口木放線菌研究会編)67頁記載の薄
層クロマトグラフィー法により分析
LL−ジアミノピメリン酸及びグリシンを含有する。以
上の菌学的性質より木菌はいずれもストレプトミセス属
に属するものと認定し、ストレプトミセス・エスピーと
命名した。2 cell wall type I5P-II agar plate medium, 50°C, cultured for 17 days, the bacterial cells were collected, hydrolyzed with 6Nl (Cβ at 100°C for 18 hours, and the supernatant synovial fluid was filtered to remove the residue). Contains LL-diaminopimelic acid and glycine analyzed by the thin-layer chromatography method described in "Identification Experimental Method" (edited by Kuchiki Actinobacteria Study Group) on page 67. Based on the above mycological properties, all woody fungi belong to the genus Streptomyces. It was recognized as belonging to the species and named Streptomyces sp.
これらの菌を培養する培地にはβ−グルコシド拮合を有
する糖を主炭素源として好ましくは唯一の炭素源として
含有せしめることによって本酵素を誘導産生させる。こ
の糖の例としてはフェニル−β−D−グルコシド、セロ
ビオース、ソホロース、ラミナリビオース、ゲンチオビ
オース、ラクトース、シュクロース、メチル−β−グル
コシド、エスクリン、アルブチン、サリシン等を挙げる
ことができる。これらのなかでフェニル−β−グルコシ
ド、セロビオース、ソホロース、メチル−β−グルコシ
ド及びエスクリンが好ましい。The enzyme is induced to be produced by containing a sugar having β-glucoside affinity as the main carbon source, preferably as the only carbon source, in the culture medium for culturing these bacteria. Examples of the sugar include phenyl-β-D-glucoside, cellobiose, sophorose, laminaribiose, gentiobiose, lactose, sucrose, methyl-β-glucoside, esculin, arbutin, salicin, and the like. Among these, phenyl-β-glucoside, cellobiose, sophorose, methyl-β-glucoside and esculin are preferred.
炭素源以外の成分はストレプトミセス属菌を培養する通
常の培地と同様でよく、ペプトン、肉エキス、麦芽エキ
ス、酵母エキス、大豆蛋白加水分解物などの有機窒素化
合物、アンモニウム塩硝酸塩等の無段窒素化合物さらに
はビタミン等の微量栄養物等を適宜含む培地を用いる。Ingredients other than the carbon source may be the same as those of ordinary media for culturing Streptomyces bacteria, such as organic nitrogen compounds such as peptone, meat extract, malt extract, yeast extract, soybean protein hydrolyzate, and ammonium salts and nitrates. A medium appropriately containing nitrogen compounds and micronutrients such as vitamins is used.
このような培地をpH4〜8程度、好ましくは中性付近
に調整して前記微生物を接種し、16〜48時間程度B
’liあるいは通気攪拌培養することにより本酵素を主
として菌体内に生成蓄積させることができる。前記2例
の微生物はいずれも好熱菌であり、培養温度は40〜5
5℃程度が適当である。Adjust the pH of such a medium to about 4 to 8, preferably around neutrality, inoculate it with the microorganisms, and leave it for about 16 to 48 hours.
The present enzyme can be mainly produced and accumulated within the bacterial cells by 'li or aeration agitation culture. Both of the microorganisms in the above two examples are thermophilic bacteria, and the culture temperature is 40-50%.
Approximately 5°C is appropriate.
本酵素を分離精製する方法としては、まず培養液から菌
体と培養液上清画分を分離し、菌体画分は超音波処理な
どによる物理的方法あるいは薬剤を用いた化学的方法で
凹体を破壊する。そして、両両分をゲル濾過、イオン交
換クロマトグラフィー、硫安塩析等公知の酵素精製法を
利用して精製を行なえばよい。The method for isolating and purifying this enzyme is to first separate the bacterial cells and the culture supernatant fraction from the culture solution, and then isolate the bacterial cell fraction by a physical method such as ultrasonication or a chemical method using drugs. destroy the body. Then, both components may be purified using known enzyme purification methods such as gel filtration, ion exchange chromatography, and ammonium sulfate salting out.
本発明の酵素はグルコースによる生成物阻害が少なく、
セロビオースを分解してグルコースを生成する反応を後
半においても効率よく進行させることができる。The enzyme of the present invention has less product inhibition by glucose;
The reaction of decomposing cellobiose to produce glucose can proceed efficiently even in the latter half.
実施例1
ストレプトミセス・エスピーAJ 9456 FERM
P−8861を下表に示す組成の寒天培地(ISP−
IT)に50℃で24hr培養した。Example 1 Streptomyces sp. AJ 9456 FERM
P-8861 was placed on an agar medium (ISP-
IT) and cultured at 50°C for 24 hours.
成分 添加量
酵母エキス 4g/l
麦芽エキス 10g/6
グルコース 4 g / 1
寒 天 15g/1
(pH7,3)
この菌体を下表に示す組成のβ−グルコシダーゼ生生産
培地50m合含む坂ロフラスコ(500ml)に接種し
、50℃で24hr振盪培養した。Ingredients Added amount Yeast extract 4 g/l Malt extract 10 g/6 Glucose 4 g/1 Agar 15 g/1 (pH 7,3) This bacterial cell was placed in a Sakaro flask containing 50 m of β-glucosidase live production medium with the composition shown in the table below ( 500ml) and cultured with shaking at 50°C for 24 hours.
成 分 添加量Bacto
Yeast Nigrogen Ba5e 6
.7 g / 1セロビオース 20
gel。Ingredient Addition amount Bacto
Yeast Nigrogen Ba5e 6
.. 7 g/1 cellobiose 20
gel.
硝酸カルシウム 3g/ff炭酸カル
シウム(別殺菌) 20g//!(pH7,0)
得られた菌体5g(湿重量)を0.2%KC1で2回洗
浄後、10m7!のO,]、 M TESにQiした。Calcium nitrate 3g/ff Calcium carbonate (separately sterilized) 20g//! (pH 7.0) After washing 5 g (wet weight) of the obtained bacterial cells twice with 0.2% KC1, 10 m7! O, ], M TES was Qi.
15°C以下の温度で音波破砕し、16.00Orpm
で1hr遠心した。得られた上清5m! (蛋白量10
0■)をIIEAE−Toyopearl 650M(
1,5X 8 Cl11)のカラムに導入した。このカ
ラムは予め0.05 M Tris・HC2緩衝液、p
H8,0で平衡化しておき、上記上清を通液した後に再
び同緩衝液30m1lをカラムに通した。引きつづき同
緩衝液100m!!と0.5MNaCj!を含む同緩衝
液100とを用いてグラジェント溶出を行ない、第6図
に示す2つの活性のピークのうち大きいピークの画分障
73〜78を集めて酵素液として使用した。尚、図中白
丸はセロビオースを基質として生成したグルコースを比
色法で測定したOD5゜、を示し、黒点はp−ニトロフ
ェニル−β−グルコースを基質として生成したp−二ト
ロフェノールを測定した○D 401)を示している。Sonically disrupted at a temperature below 15°C, 16.00Orpm
The mixture was centrifuged for 1 hour. Obtained supernatant 5m! (Protein amount 10
0 ■) to IIEAE-Toyopearl 650M (
1,5× 8 Cl11) column. This column was prepared in advance with 0.05 M Tris·HC2 buffer, p
Equilibration was carried out with H8.0, and after the above supernatant was passed through the column, 30 ml of the same buffer solution was passed through the column again. Continue with 100m of the same buffer! ! and 0.5MNaCj! Gradient elution was carried out using the same buffer solution 100 containing 100% of the total activity, and fractions 73 to 78, which had the larger peak of the two activity peaks shown in FIG. 6, were collected and used as an enzyme solution. In the figure, white circles indicate OD5°, which was measured by colorimetry of glucose produced using cellobiose as a substrate, and black dots indicate p-nitrophenol produced using p-nitrophenyl-β-glucose as a substrate. D401).
図中の斜線はNaC1濃度を示している。The diagonal line in the figure indicates the NaCl concentration.
0、1 M MES緩衝液pH6,5,9■セロビオー
スと上記の酵素液0.05mff1(蛋白質50mg)
を含む全容0,2mlを50゛Cで1時間インキユヘー
トした。生成物を3層クロマト(ブタノール:ピリジン
:水=6:4:3)法によって同定した結果、グルコー
スに相当したRf値を示すスポットが検出された。この
生成グルコース量を、グルコースオキシターゼ法(グル
コース−〇−テスト、和光純薬■製)を用いて定量した
結果5■のグルコースが生成していた。次にセロビオー
スの代りにp−ニトロフェニルーβ−グルコサイド0.
3■を添加して50°C110分インキュベートしたと
ころ0.1■のグルコース及び0.077■のp−ニト
ロフェノールが生成していた。0, 1 M MES buffer pH 6, 5, 9 ■ Cellobiose and the above enzyme solution 0.05 mff1 (50 mg protein)
A total volume of 0.2 ml containing the solution was incubated at 50°C for 1 hour. As a result of identifying the product by three-layer chromatography (butanol:pyridine:water=6:4:3), a spot with an Rf value corresponding to glucose was detected. The amount of glucose produced was quantified using the glucose oxidase method (Glucose-〇-Test, manufactured by Wako Pure Chemical Industries, Ltd.), and it was found that 5㎜ of glucose was produced. Next, instead of cellobiose, p-nitrophenyl-β-glucoside 0.
When 3.5 μm was added and incubated at 50° C. for 110 minutes, 0.1 μg of glucose and 0.077 μm of p-nitrophenol were produced.
ワットマンセルロース粉末CC41200■、0.1M
#酸緩衝液、pH5,0、スポロトリウムセルロフィル
ムへTCC20493(AJ 6987)のセルラーゼ
(蛋白20■)、及び前述のβ−グルコシダーゼの酵素
液2 ml (蛋白2■)を含む全容量10m7!の反
応液を50℃で1時間攪拌しながらインキュベートした
後、生成グルコースを定量したところ下表に示す結果が
得られた。Whatman cellulose powder CC41200■, 0.1M
#Acid buffer, pH 5.0, total volume 10m7 containing TCC20493 (AJ 6987) cellulase (20μ protein) and 2 ml (2μ protein) of the aforementioned β-glucosidase enzyme solution! After incubating the reaction solution at 50°C for 1 hour with stirring, the produced glucose was quantified, and the results shown in the table below were obtained.
酵 素 生成グルコース(■)セルラーゼ
34
セルラーゼ+“β−グルコシダーゼ” 51実施例
2
ストレプトミセス・エスピーAJ 9457を実施例1
と同様に培養し、得られた菌体から酵素液を得た。Enzyme Produces glucose (■) Cellulase 34 Cellulase + “β-glucosidase” 51 Example 2 Streptomyces sp. AJ 9457 Example 1
The cells were cultured in the same manner as above, and an enzyme solution was obtained from the resulting bacterial cells.
本発明の酵素を用いることによりセルロースから酵素法
でグルコースを効率よくかつ高収率で取得することがで
きる。By using the enzyme of the present invention, glucose can be obtained efficiently and in high yield from cellulose by an enzymatic method.
第1図及び第2図はいずれも本酵素の至適pHを示す曲
線の図であり、第3図は安定pH範囲をそして第4図は
熱安定性をそれぞれ示す曲線の図である。第5図は本酵
素と従来の酵素についてグルコース濃度と活性の関係を
示す曲線の図である。
第6図は本酵素をイオン交換クロマトグラフィーで精製
した際の溶離曲線を示す図である。
特許出願人 新燃料油開発技(、トj研究組合代理人
弁理士 1)中 政 浩 はか1名ViAiz5.l、
、ヒ’ l’i ’(’i’ を二 −しパ1−)第1
図
pH
第2図
04↓
□
pH
第 314
pH
第4図
8− て (°C)
第5図
7ILフ一人 (Ml
第6図
手 続 補 正 肉 〈自発)
13!1f4161り丁9月8[1
特許庁艮官 黒 1)l′f1 雄 殿1事nの表示
特願昭61−170867号
2)明の名称
β−グルコシダーぜ及びその製TiF去3補正をする者
事件との関係 特許出願人
名称 新燃料油開発技術研究組合
4代 理 人
居所 〒104東京都中央区八丁堀三丁目2)番3−6
07号電話(03)555−0022
5補正の1唖
図面
6補正の内容Both FIGS. 1 and 2 are curves showing the optimum pH of the enzyme, FIG. 3 is a curve showing the stable pH range, and FIG. 4 is a curve showing the thermostability. FIG. 5 is a graph showing the relationship between glucose concentration and activity for the present enzyme and a conventional enzyme. FIG. 6 is a diagram showing an elution curve when this enzyme was purified by ion exchange chromatography. Patent applicant New fuel oil development technology (Toj research association agent)
Patent attorney 1) Masahiro Naka 1 person ViAiz5. l,
, hi'l'i'('i' is two - and pa1-) 1st
Figure pH Figure 2 04 ↓ □ pH No. 314 pH Figure 4 8- (°C) Figure 5 7IL F alone (Ml Figure 6 Procedure Correction Meat <Spontaneous) 13! 1f4161Right September 8 [ 1 Patent Office Officer Black 1) l'f1 Male Indication Patent Application No. 170867/1983 2) Relationship with the Ming Dynasty's Name β-Glucosidase and its Production TiF 3 Amendment Case Patent Application Name Osamu, 4th generation New Fuel Oil Development Technology Research Association Address 3-3-6 Hatchobori 3-2-2, Chuo-ku, Tokyo 104
Telephone No. 07 (03) 555-0022 1 page of 5 amendments Contents of drawing 6 amendments
Claims (2)
在時の65%以上であるβ−グルコシダーゼ(1) β-glucosidase whose relative activity in the presence of 1M glucose is 65% or more of that in the absence of glucose
在時の65%以上であるβ−グルコシダーゼを産生しう
る能力を有するストレプトミセス属微生物をβ−グルコ
シダーゼ生産培地で培養して培養液中に該β−グルコシ
ダーゼを生成蓄積せしめ、これを採取することを特徴と
する1Mグルコース存在下の相対活性がグルコース不在
時の65%以上であるβ−グルコシダーゼの製造法(2) Streptomyces microorganisms capable of producing β-glucosidase whose relative activity in the presence of 1M glucose is 65% or more of that in the absence of glucose are cultured in a β-glucosidase production medium, and the β-glucosidase is added to the culture solution. - A method for producing β-glucosidase whose relative activity in the presence of 1M glucose is 65% or more of that in the absence of glucose, which comprises producing and accumulating glucosidase and collecting it.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP17086786A JPS6328389A (en) | 1986-07-22 | 1986-07-22 | Beta-glucosidase and production thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP17086786A JPS6328389A (en) | 1986-07-22 | 1986-07-22 | Beta-glucosidase and production thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS6328389A true JPS6328389A (en) | 1988-02-06 |
Family
ID=15912783
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP17086786A Pending JPS6328389A (en) | 1986-07-22 | 1986-07-22 | Beta-glucosidase and production thereof |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6328389A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2706907A1 (en) * | 1993-06-21 | 1994-12-30 | Ifremer | Beta-glucosidase, microorganism producing it and process for obtaining it |
CN103114100A (en) * | 2013-02-27 | 2013-05-22 | 广西科学院 | Beta-glucosaccharase gene S-bgl3 and application thereof |
-
1986
- 1986-07-22 JP JP17086786A patent/JPS6328389A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2706907A1 (en) * | 1993-06-21 | 1994-12-30 | Ifremer | Beta-glucosidase, microorganism producing it and process for obtaining it |
CN103114100A (en) * | 2013-02-27 | 2013-05-22 | 广西科学院 | Beta-glucosaccharase gene S-bgl3 and application thereof |
CN103114100B (en) * | 2013-02-27 | 2014-08-13 | 广西科学院 | Beta-glucosaccharase gene S-bgl3 and application thereof |
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