JPH0257917B2 - - Google Patents
Info
- Publication number
- JPH0257917B2 JPH0257917B2 JP58162960A JP16296083A JPH0257917B2 JP H0257917 B2 JPH0257917 B2 JP H0257917B2 JP 58162960 A JP58162960 A JP 58162960A JP 16296083 A JP16296083 A JP 16296083A JP H0257917 B2 JPH0257917 B2 JP H0257917B2
- Authority
- JP
- Japan
- Prior art keywords
- chitobiase
- chitin
- minutes
- nitrophenyl
- medium
- 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
- 229920002101 Chitin Polymers 0.000 claims description 26
- 108010055851 Acetylglucosaminidase Proteins 0.000 claims description 25
- 102100036495 Di-N-acetylchitobiase Human genes 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 20
- 102000012286 Chitinases Human genes 0.000 claims description 9
- 108010022172 Chitinases Proteins 0.000 claims description 9
- OMRLTNCLYHKQCK-DHGKCCLASA-N 4-nitrophenyl N-acetyl-beta-D-glucosaminide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 OMRLTNCLYHKQCK-DHGKCCLASA-N 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 241000607528 Aeromonas hydrophila Species 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 22
- 239000000243 solution Substances 0.000 description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 19
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 239000002609 medium Substances 0.000 description 15
- 238000000034 method Methods 0.000 description 11
- 239000011780 sodium chloride Substances 0.000 description 11
- 229920001817 Agar Polymers 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- MBLBDJOUHNCFQT-LXGUWJNJSA-N aldehydo-N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 9
- 239000001888 Peptone Substances 0.000 description 8
- 108010080698 Peptones Proteins 0.000 description 8
- 241000607598 Vibrio Species 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 235000019319 peptone Nutrition 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000005185 salting out Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000008399 tap water Substances 0.000 description 5
- 235000020679 tap water Nutrition 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000001794 chitinolytic effect Effects 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- LIVXWXAMTVJGCO-UHFFFAOYSA-N 2,4-diamino-6,7-diisopropylpteridine Chemical compound NC1=NC(N)=C2N=C(C(C)C)C(C(C)C)=NC2=N1 LIVXWXAMTVJGCO-UHFFFAOYSA-N 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 241000588881 Chromobacterium Species 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006114 decarboxylation reaction Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 229940050410 gluconate Drugs 0.000 description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 229950006780 n-acetylglucosamine Drugs 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- DGZSVBBLLGZHSF-UHFFFAOYSA-N 4,4-diethylpiperidine Chemical compound CCC1(CC)CCNCC1 DGZSVBBLLGZHSF-UHFFFAOYSA-N 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 238000002238 CM-cellulose chromatography Methods 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
- 108010076119 Caseins Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 1
- 108050008072 Cytochrome c oxidase subunit IV Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 241000607594 Vibrio alginolyticus Species 0.000 description 1
- 241000607493 Vibrionaceae Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 108010077311 arginine oxidase Proteins 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
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- 210000003495 flagella Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
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- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229940049906 glutamate Drugs 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
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- 238000007747 plating Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
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- 150000008163 sugars Chemical class 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
Description
本発明は新規なキトビアーゼの製造法に関す
る。
本発明者らは天然の土壤より数多くの微生物を
単離し、その生産物について種々研究を行つた結
果、今回本発明者によつて分離された細菌がキチ
ン分解酵素を多量に産生することを見出し、本発
明を完成した。
すなわち、本発明は新規なキトビアーゼの製造
法を提供するものである。
本発明のキトビアーゼを産生する細菌は次のよ
うな菌学的性質を有する。
(1) 形態
直状、球形末端を有する桿形、大きさ1.0〜
2.4μｍ、運動性あり、極単毛性で鞭毛を有す
る、グラム陰性
(2) 生育状態
0.2％コロイドキチン、0.1％ペプトン、0.1
％肉汁エキス、0.3％塩化ナトリウム及び2.0
％寒天含有（PH7.0）キチン−寒天平板培地
中、30℃で72時間インキユベーシヨンする
と、明確なコロニーを形成する。
肉汁液体培地中、37℃で生育する。
7.5％塩化ナトリウム含有肉汁液体培地中
で生育しない。
単一窒素源としてアンモニア、単一炭素源
としてグルコース、Ｌ−アルギニン、Ｌ−ア
スパラギン、Ｌ−ヒスチジン、Ｌ−グルタミ
ン酸、Ｌ−セリン又はＬ−アラニンを含有す
る無機培地中で生育する。
トリプチケースダイズ寒天培地上で褐色の
水溶性色素を生成しない。
アルギニン、アスパラギン、ロイシン及び
メチオニンを含有する混合培地中で生育す
る。
(3) 生理学的性質
硝酸塩の還元：陽性
Ｖ−Ｐテスト：陰性
インドールの生成：陽性（0.1％トリプト
フアン含有トリプトンブロス中）
硫化水素の生成：陽性（2.5％ペプトン水
中）
デンプンの加水分解：陽性
ウレアーゼ：陰性
オキシダーゼ：陽性
カタラーゼ：陽性
チトクローム オキシダーゼ：陽性
生育の温度範囲：13〜42℃で生育し、27〜
30℃において最もよく生育
生育のPH範囲：5.5〜9.0
酸素に対する態度：通性嫌気性
糖類に対する資化性、酸およびガスの生成
（資化性、酸およびガスの生成があるもの、
ないものを、それぞれ＋、−で示す）：
The present invention relates to a novel method for producing chitobiase. The present inventors isolated a large number of microorganisms from natural soil and conducted various studies on their products. As a result, the present inventors discovered that the bacteria isolated by the present inventors produced large amounts of chitinolytic enzymes. , completed the invention. That is, the present invention provides a novel method for producing chitobiase. Bacteria producing chitobiase of the present invention have the following mycological properties. (1) Morphology Straight, rod-shaped with a spherical end, size 1.0~
2.4 μm, motile, extremely monociliated, with flagella, Gram negative (2) Growth status 0.2% colloidal chitin, 0.1% peptone, 0.1
% meat juice extract, 0.3% sodium chloride and 2.0%
After incubation for 72 hours at 30°C in chitin-agar plates containing % agar (PH 7.0), distinct colonies are formed. Grows at 37°C in broth liquid medium. Does not grow in broth liquid medium containing 7.5% sodium chloride. Grow in mineral media containing ammonia as the sole nitrogen source and glucose, L-arginine, L-asparagine, L-histidine, L-glutamic acid, L-serine or L-alanine as the sole carbon source. Does not produce brown water-soluble pigment on trypticase soy agar. It grows in a mixed medium containing arginine, asparagine, leucine and methionine. (3) Physiological properties Nitrate reduction: Positive V-P test: Negative Indole formation: Positive (in tryptone broth containing 0.1% tryptophan) Hydrogen sulfide formation: Positive (in 2.5% peptone water) Starch hydrolysis: Positive Urease: Negative Oxidase: Positive Catalase: Positive Cytochrome Oxidase: Positive Growth temperature range: Grows at 13-42℃, 27-42℃
Grows best at 30℃ Growth PH range: 5.5-9.0 Attitude towards oxygen: Facultative anaerobic Assimilation of sugars, production of acids and gases (those with assimilation, production of acids and gases,
Items that are not available are indicated by + and -, respectively):
【表】【table】
【表】
(4) その他の性質
グルコン酸の酸化：陰性（グルコン酸オキ
シダーゼ試験）
リジンの脱炭酸反応：陰性〔モラKg
（Moller）法〕
塩化ナトリウムの耐性：陽性（1.0％NaCl
含有ブロスにおいて生育最大）
シアン化カリウムの耐性：陽性（モラー
法）
フオスフアターゼ：陽性
カゼインの加水分解：陽性
ゼラチン溶解性：陽性
デオキシリボヌレアーゼ：陽性
リボヌクレアーゼ：陽性
アルギニン脱水素酵素：陽性
グルタミン酸の脱炭酸反応：陰性
ビブリオスタテイツク試薬、２，４−ジア
ミノ−６，７−ジイソプロプル プテリジン
（０／129）に対する反応性：陰性
２，３−ブタンジオールからアセトインが
生産されるが、グルコースからは生産されな
い。
ブキヤナン（Buchanan、P.）等（1974年）、
コワン（Cowan、S.T.）（1974年）、及びゲルハ
ルト（Gerhardt、P.）等（1981年）の系統的方
法により調べられた上記性状と本発明に係るキチ
ン溶解性に基づき、本細菌の種属を検索すると、
キチン分解性を有する生物としては、コワン
（1974年）のビブリオ（ベネツケア）・パラヘモリ
テイカス〔Viibrio（Beneckea）
parahemolyticus〕及びバウマン（Bauman、P.
L.）等のビブリオ（クロモバクテリウム）・アル
ギノリテイカス（V.（Chromobacterium）
alginolyticus〕が認められる〔ブキヤナン等
（1974）によれば、この２種の生物はビブリオ・
パラヘモリテイカスに分類されている〕。キチン
溶解性の細菌は既に報告があり、それらはセラテ
イア（Serratia）属〔モンリアル（Monreal、
J.）等（1969年）、レイド（Reid、J.D）等（1981
年）〕、ビブリオ属〔内田等（1979年）〕、ベネツケ
ア属〔高橋等（1982年）〕及びストレプトミセス
属〔レイノルズ（Reynolds、D.M.）（1954年）〕
であると記載されている〔ブキヤナン等（1974
年）に依ればベネツケアはビブリオ属に属すると
考えられている〕。これらを参考にすると、本細
菌はビブリオナセエ科に属するものと考えられ
る。因みに、本細菌はペプトンから硫化水素を生
成する能力があり、またビブリオスタテイツク
（Vibriostatic）試薬０／129に感作しない点でビ
ブリオ属とは全く相違する。
叙上の証拠より、本細菌は菌株アエロモナス・
ヒドロフイラ・亜アネロゲネス ATCC15467
（IFO13282）に類似する。なぜなら、本細菌はＶ
−Ｐ反応、グルコン酸オキシダーゼ試験が陰性で
あり、グリセリン及びグルコースからガスを生成
しない〔ブキヤナン等（1974年）〕。然し、本細菌
が強いキチン溶解活性を有するのに対し、上記菌
株はキチン溶解性を全く示さない点で全く相違す
る。そこで本発明者は、本細菌を公知の菌株と区
別するために、アエロモナス・ヒドロフイラ・亜
アネロゲネス A52（Aeromonas hydrophila
subsp.anaerogenesA52；以下において細菌52と
略称することがある）と命名し、工業技術院微生
物工業技術研究所に受託番号微工研菌寄7206号
（FERM Ｐ−7206）として寄託した。
本細菌は次の如くして分離、純化される。分離
は、0.2％コロイドキチン、0.1％ペプトン、0.1％
肉汁エキス、0.3％塩化ナトリウム及び2.0％寒天
含有キチン−寒天平板培地（PH7.0）で、試料の
懸濁液を１白金耳量画線する（画線平板法）こと
により行つた。30℃で72時間インキユベーシヨン
後、コロニーを採取し、上記と同組成の斜面キチ
ン−寒天培地に保存する。コロニーの周囲にはコ
ロイドキチンが溶解している明確な領域が形成さ
れる。分離したコロニーからの当該細菌の純化は
キチン−寒天培地及び普通ブロス−寒天培地上で
交互に６回平板培養することにより行う。最後に
47個の分離物のうち、キチン−寒天培地上で生育
が早く、大きく明確なコロニーを形成するものを
細菌A52として選択した。
斯くして得られた細菌は、1.0％肉汁エキス、
1.0％ペプトン、0.5％塩化ナトリウム、2.0％寒天
含有普通ブロス−傾斜培地（1N−NaOHでPH7.0
に調整）中、28℃で３日間培養した後室温で存在
し、１箇月ごとに新しい培地に植え継ぎ保存す
る。
本発明のキトビアーゼは、上記細菌を栄養源培
地に接種し培養せしめることにより製造される。
培養に用いられる培地としては、酵素誘導基質で
あるキチンと当該菌が利用する栄養源を含むもの
であれば何れでもよいが、例えば1.0％エビ殻キ
チン、0.2％ブドウ糖、0.5％ペプトン、1.0％酵母
エキス、0.7ｇ／リン酸二水素カリウム、0.3％
塩化ナトリウムを含有（1N−NaOHでPH7.0に調
整）するものが挙げられる。
培養法としては、振盪培養が好適である。培養
に適当な温度は25〜30℃であるが多くの場合28℃
付近で培養する。２〜３日間培養後、培養液は次
の操作に付される。
キトビアーゼの単離は、後記実施例に示す如
く、キトビアーゼの理化学的性状を考慮して種々
の方法を適当に組合せることによつて行う。
すなわち、キトビアーゼは通常、培養液中に
存在するので、遠心分離又は過等の手段によつ
て培養物から細菌を分離した後、培養液に硫酸
アンモニウムを添加して塩析を行う。次いで塩析
により析出したタンパクの沈澱を0.1Mトリス−
塩酸緩衝液（PH7.0）に溶かし、これを遠心分離
してその上澄液を粗酵素液とする。粗酵素液はキ
トビアーゼ及びキチナーゼを含有する。
粗酵素液からキトビアーゼの単離は、キトビア
ーゼ及びキチナーゼのコロイドキチンへの吸着性
の相違を利用して行う。すなわち、粗酵素液及び
コロイドキチンをトリス−塩酸緩衝液と混ぜ、キ
チナーゼをコロイドキチンに吸着させた後遠心分
離し上清を得、これをキトビアーゼ画分とする。
なお、キチナーゼは遠心分離により得られた沈澱
から得られる。
キトビアーゼ画分から、キトビアーゼの分離精
製は、CM−セルロースクロマトグラフイー及び
DEAE−セフアデツクスＡ−50によるカラムクロ
マトグラフイーを利用して行なわれる。
以上の如くして得られたキトビアーゼは次のよ
うな理化学的性質を有する。
作用：キチナーゼと共に作用してキチンを分
解する。
至適PH：PH7.0
Km*：ｐ−ニトロフエニル−Ｎ−アセチル
グルコサミニド0.17ｍＭ
PH安定性：37℃で30分処理した場合、PH6.0
〜PH9.0において90％以上の残存活性を示す。
至適温度：PH7.0において、ｐ−ニトロフエ
ニル−Ｎ−アセチルグルコサミニドを基質とし
た場合50℃付近にある。
温度安定性：ｐ−ニトロフエニル−Ｎ−アセ
チルグルコサミニド基質で、PH7.0において、
０〜40℃、30分処理して80％以上の残存活性を
示す。
等電点**：PH6.8付近
分子量***：115000
＊〔Km値の測定〕
5.0ｍＭ ｐ−ニトロフエニル−Ｎ−アセチル
グルコサミニド0.2ml、50mMトリス−マレイン
酸緩衝液（PH7.0）0.7ml、及び酵素液0.1mlからな
る反応液を37℃で10分間インキユベートする。次
いで、この反応液に0.25M炭酸ナトリウム溶液
2.0mlを加え反応を停止し、同時にｐ−ニトロフ
エノールの発色を起こさせた。生成したＮ−アセ
チルグルコサミンはｐ−ニトロフエノールと等モ
ルであるから、405nｍの吸光度を測り、ｐ−ニ
トロフエノールの検量線からその量を求めた。キ
トビアーゼ活性は、ｐ−ニトロフエニル−Ｎ−ア
セチルコサミニドから１分間に1μmolのＮ−アセ
チルグルコサミンを生成する酵素量を１単位とし
た。結果はラインウイーバー−バーク
（Lineweaver−Burk）プロツトからKm値を求
めた。なお、酵素液は精製キトビアーゼ標品を用
い、１反応液中の酵素量は0.054Uで行つた。
＊＊〔等電点電気泳動〕
ポリアクリルアミドゲルデイスク等電点電気泳
動はデイビス、ビー．ジエー．Ann.N.Y.Acad.
Sci.、121、404（1964）に基き、7.5％ゲル、トリ
ス−グリシン緩衝液（PH8.4）中で行つた。チユ
ーブ一本あたり２〜４ｍＡの電流を流し、５℃で
泳動した。
＊＊＊〔分子量〕
SDS−ポリアクリルアミドゲル電気泳動法によ
る。
斯くして得られる本発明のキトビアーゼは細胞
壁溶解酵素としてプロトプラストの形成等に利用
できるものである。
次に実施例及び参考例を挙げて説明する。
参考例 １
(1) エビ殻キチンの調製
冷凍エビ殻を解凍し、10分間ワーリングブレ
ンダー処理を行ない、水道水で３回以上水洗す
る。得られたエビ殻フレークを、1N水酸化ナ
トリウムに１晩浸漬して除タンパクを行ない、
水道水で３回以上水洗する。次いで得られた除
タンパクエビ殻フレークを1N塩酸に１晩浸漬
してカルシウム分を除く。以上の操作により得
られた精製エビ殻フレークを水道水で水洗後、
1N水酸化ナトリウムでPH7.0に調整後、10分間
ワーリングブレンダー処理し、乾物量を２％に
調整して、オートクレーブで120℃にて20分間
滅菌する。
(2) コロイドキチンの調製
(1)で得たエビ殻キチンをボールミルで約24時
間粉砕しボールミルキチンとした。このボール
ミルキチンを以下の操作に付しコロイドキチン
を得た。
ａ 冷却した乳鉢をアセトンで湿らせ、ボール
ミルキチンと濃塩酸をよく混合する。
ｂ 大量の冷水中によく撹拌しながら滴下分散
させる。
ｃ 18000ｇで10分間遠心分離することにより
水洗する。
ｄ ワーリングブレンダーで10分間処理する。
ｅ 18000ｇで10分間遠心分離してコロイドキ
チンを集め、更に0.025Mトリス−塩酸緩衝
液（PH7.2）で洗う。
斯くして得られたコロイドキチンは、適宜緩衝
液に分散させ使用に供される。
実施例 １
(1) 酵素生産のための培養
水道水１にペプトン15ｇ、酵母エキス５
ｇ、リン酸二水素カリウム0.68ｇを加え、1N
−水酸化ナトリウムでPH7.0に調整した前培養
培地を、綿栓試験管に５mlずつ入れ、常法に従
い滅菌する。次いでこれに保存培地から細菌
A52を一白金耳接種し、28℃で24時間振盪培養
を行なう。本培養は、水道水１に、エビ殻キ
チン10ｇ、グルコース２ｇ、ペプトン５ｇ、酵
母エキス10ｇ、リン酸二水素カリウム0.68ｇ、
塩化ナトリウム３ｇを加え、1N−水酸化ナト
リウムでPH7.0に調整した本培養培地を、500ml
容フラスコに70mlずつ分注し、常法に従い滅菌
する。次いで、これに培養終了後の前培養培地
１mlを接種し、28℃で72時間振盪培養
（240rpm）した。
(2) 粗酵素液の調製
培養終了後、本培養培地から18000ｇで20分
間遠心分離することにより菌体を除いた後、細
菌による汚染を防ぐために最終濃度0.02％とな
るようにアジ化ナトリウムを加え培養液とし
た。次いで０℃冷却下、スターラーで静かに撹
拌しながら、培養液に80％飽和になるように
固形硫安を徐々に加え塩析を行つた。このと
き、培養液のPHが酸性側に傾かないように、
1N−水酸化ナトリウムでPH7.0付近に保持しな
がら行つた。塩析は０〜４℃に冷却しながら１
時間以上行つた。塩析により析出したタンパク
の沈澱は、18000ｇで20分間遠心分離して集め
た。この沈澱を培養液の10分の１容の0.1M
トリス−塩酸緩衝液（PH7.0）に溶かし、０〜
４に冷却して１時間静置後、不溶物を18000ｇ
で20分間遠心分離で除き、上清を粗酵素液と
し、−20℃で凍結保存した。
(3) キチン吸着によるキチナーゼとキトビアーゼ
の分離
コロイドキチンに対する両酵素の親和力の違
いにより次の如くして分離した。
粗酵素液75ml（キチナーゼ約887U）と、コ
ロイドキチン2.5ｇ（乾物重量）を含む25ｍＭ
トリス−塩酸緩衝液（PH7.2）300mlを混ぜ、
時々撹拌しながら氷冷下で１時間、キチナーゼ
をコロイドキチンに吸着させた。次いで18000
ｇで20分間遠心分離を行ない、上清をキトビア
ーゼ画分とした。
(4) キトビアーゼの精製
実施例１の(3)で得られたキトビナーゼ画分を
１ｍＭ PMSFの存在下でエバポレータ濃縮
後、５ｍＭトリス−塩酸緩衝液（PH7.2）に対
して一晩透析した標品を、50ｍＭ酢酸緩衝液
（PH5.2）で平衡化したCM−セルロースのカラ
ム（2.6×45cm）でイオン交換した。なお、溶
出は塩化ナトリウム濃度勾配０→0.3Mで、流
速は40ml／時とした。次いで、溶出液を限外
過により濃縮・脱塩した後、0.05M塩化ナトリ
ウム含有25ｍＭトリス−塩酸緩衝液（PH7.2）
で平衡化させたDEAE−セフアデツクスＡ−50
のカラムクロマトグラムに付し（カラムサイズ
2.6×45cm）精製キトビアーゼを得た。なお、
溶出は塩化ナトリウム濃度勾配0.05→0.2Mで、
流速は44ml／時とした。
以上の各操作段階におけるトキビアーゼの精製
度及び回収率を第１表に、またDEAE−セフアデ
ツクスＡ−50によるカラムクロマトグラムを第１
図に示す。第１表より明らかな如く、上記操作に
より粗酵素液中のキトビアーゼは144培に精製さ
れた。[Table] (4) Other properties Gluconic acid oxidation: Negative (gluconate oxidase test) Lysine decarboxylation reaction: Negative [Mora Kg
(Moller) method] Sodium chloride resistance: Positive (1.0% NaCl
Potassium cyanide resistance: Positive (Molar method) Phosphatase: Positive Casein hydrolysis: Positive Gelatin solubility: Positive Deoxyribonurease: Positive Ribonuclease: Positive Arginine dehydrogenase: Positive Glutamate decarboxylation: Negative Vibriostatic reagent, 2,4-diamino-6,7-diisopropyl Pteridine (0/129) Reactivity: Negative Acetoin is produced from 2,3-butanediol, but not from glucose. Buchanan, P. et al. (1974),
Based on the above properties investigated by the systematic method of Cowan, ST. (1974) and Gerhardt, P. et al. (1981) and the chitin solubility according to the present invention, When you search for
An example of an organism capable of decomposing chitin is Vibrio (Beneckea) parahaemolyticus [Vibrio (Beneckea)] by Cowan (1974).
parahemolyticus] and Bauman (P.
Vibrio (Chromobacterium) such as L.) and V. (Chromobacterium)
[According to Bukiyanan et al. (1974), these two species are Vibrio alginolyticus].
It is classified as Parahaemolyticus]. Chitinolytic bacteria have already been reported, and they belong to the Serratia genus [Monreal,
J.) et al. (1969), Reid, J.D. et al. (1981)
)], Vibrio [Uchida et al. (1979)], Venetskea [Takahashi et al. (1982)], and Streptomyces [Reynolds, DM (1954)].
[Bukiyanan et al. (1974)
According to the 2011 study, Venetcea is considered to belong to the genus Vibrio]. Based on these, this bacterium is considered to belong to the family Vibrionaceae. Incidentally, this bacterium is completely different from the genus Vibrio in that it has the ability to produce hydrogen sulfide from peptone and is not sensitized to Vibriostatic reagent 0/129. Based on the evidence presented above, this bacterium is a strain of Aeromonas.
Hydrophila subanerogenes ATCC15467
Similar to (IFO13282). This is because this bacterium is V
-P reaction, gluconate oxidase test is negative, and no gas is produced from glycerin and glucose [Bukiyanan et al. (1974)]. However, while this bacterium has a strong chitinolytic activity, the above-mentioned strain is completely different in that it does not show any chitinolytic activity. Therefore, in order to distinguish this bacterium from known strains, the present inventors investigated Aeromonas hydrophila A52 (Aeromonas hydrophila subanaerogenes).
subsp.anaerogenesA52 (hereinafter sometimes abbreviated as bacterium 52) and was deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession number FERM P-7206. This bacterium is isolated and purified as follows. Separation: 0.2% colloidal chitin, 0.1% peptone, 0.1%
The test was carried out by streaking one platinum loop of the sample suspension on a chitin-agar plate medium (PH7.0) containing meat juice extract, 0.3% sodium chloride, and 2.0% agar (streaking plate method). After incubation at 30° C. for 72 hours, colonies are picked and stored on slanted chitin-agar medium with the same composition as above. A distinct area of dissolved colloidal chitin forms around the colony. Purification of the bacteria from isolated colonies is carried out by plating six times alternately on chitin-agar and ordinary broth-agar. lastly
Among the 47 isolates, those that grew quickly and formed large, well-defined colonies on chitin-agar medium were selected as bacteria A52. The bacteria thus obtained were extracted from 1.0% meat juice extract,
Ordinary broth-slant medium containing 1.0% peptone, 0.5% sodium chloride, 2.0% agar (PH7.0 with 1N-NaOH)
After culturing at 28°C for 3 days in a medium (adjusted to 100°C), the cells were kept at room temperature and transferred to a new medium every month for storage. The chitobiase of the present invention is produced by inoculating the above-mentioned bacterium into a nutrient medium and culturing it.
The culture medium may be any medium as long as it contains chitin, which is an enzyme-inducing substrate, and a nutrient source used by the bacteria, such as 1.0% shrimp shell chitin, 0.2% glucose, 0.5% peptone, or 1.0%. Yeast extract, 0.7g/potassium dihydrogen phosphate, 0.3%
Examples include those containing sodium chloride (adjusted to pH 7.0 with 1N-NaOH). As a culture method, shaking culture is suitable. The appropriate temperature for culturing is 25-30℃, but in most cases it is 28℃.
Cultivate nearby. After culturing for 2 to 3 days, the culture solution is subjected to the following operation. Chitobiase is isolated by appropriately combining various methods in consideration of the physicochemical properties of chitobiase, as shown in Examples below. That is, since chitobiase is usually present in the culture solution, after separating bacteria from the culture by centrifugation or other means, ammonium sulfate is added to the culture solution to perform salting out. Next, the protein precipitate precipitated by salting out was mixed with 0.1M Tris.
Dissolve in hydrochloric acid buffer (PH7.0), centrifuge, and use the supernatant as the crude enzyme solution. The crude enzyme solution contains chitobiase and chitinase. Chitobiase is isolated from the crude enzyme solution by utilizing the difference in adsorption properties of chitobiase and chitinase to colloidal chitin. That is, the crude enzyme solution and colloidal chitin are mixed with a Tris-HCl buffer solution, chitinase is adsorbed to the colloidal chitin, and then centrifuged to obtain a supernatant, which is used as the chitobiase fraction.
Note that chitinase is obtained from the precipitate obtained by centrifugation. Chitobiase is separated and purified from the chitobiase fraction by CM-cellulose chromatography and
DEAE-Sephadex A-50 column chromatography is used. The chitobiase obtained as described above has the following physicochemical properties. Action: Works together with chitinase to degrade chitin. Optimal PH: PH7.0 Km * : p-nitrophenyl-N-acetylglucosaminide 0.17mM PH stability: PH6.0 when treated at 37℃ for 30 minutes
Shows residual activity of 90% or more at ~PH9.0. Optimum temperature: At pH 7.0, when p-nitrophenyl-N-acetylglucosaminide is used as a substrate, it is around 50°C. Temperature stability: p-nitrophenyl-N-acetylglucosaminide substrate at pH 7.0;
Shows residual activity of 80% or more after treatment at 0-40°C for 30 minutes. Isoelectric point ** : Around PH6.8 Molecular weight *** : 115000 * [Measurement of Km value] 5.0mM p-nitrophenyl-N-acetylglucosaminide 0.2ml, 50mM Tris-maleic acid buffer (PH7.0) A reaction solution consisting of 0.7 ml of enzyme solution and 0.1 ml of enzyme solution is incubated at 37°C for 10 minutes. Next, 0.25M sodium carbonate solution was added to this reaction solution.
2.0 ml was added to stop the reaction, and at the same time color development of p-nitrophenol occurred. Since the generated N-acetylglucosamine is equimolar to p-nitrophenol, the absorbance at 405 nm was measured and the amount was determined from the calibration curve of p-nitrophenol. For chitobiase activity, one unit was defined as the amount of enzyme that produced 1 μmol of N-acetylglucosamine from p-nitrophenyl-N-acetylcosaminide per minute. The Km value was obtained from the Lineweaver-Burk plot. A purified chitobiase preparation was used as the enzyme solution, and the amount of enzyme in one reaction solution was 0.054 U. ** [Isoelectric focusing] Polyacrylamide gel disk isoelectric focusing is performed by Davis, B. J.A. Ann.NYAcad.
Sci., 121 , 404 (1964) in 7.5% gel, Tris-glycine buffer (PH8.4). A current of 2 to 4 mA was applied to each tube, and electrophoresis was performed at 5°C. *** [Molecular weight] Based on SDS-polyacrylamide gel electrophoresis. The chitobiase of the present invention thus obtained can be used as a cell wall lytic enzyme for the formation of protoplasts, etc. Next, examples and reference examples will be given and explained. Reference Example 1 (1) Preparation of shrimp shell chitin Thaw frozen shrimp shells, process in a Waring blender for 10 minutes, and wash with tap water at least 3 times. The obtained shrimp shell flakes were soaked in 1N sodium hydroxide overnight to remove protein.
Rinse at least 3 times with tap water. Next, the obtained deproteinized shrimp shell flakes are soaked in 1N hydrochloric acid overnight to remove calcium. After washing the purified shrimp shell flakes obtained by the above procedure with tap water,
After adjusting the pH to 7.0 with 1N sodium hydroxide, process in a Waring blender for 10 minutes, adjust the dry matter amount to 2%, and sterilize in an autoclave at 120°C for 20 minutes. (2) Preparation of colloidal chitin The shrimp shell chitin obtained in (1) was ground in a ball mill for about 24 hours to obtain ball milled chitin. This ball milk chitin was subjected to the following operations to obtain colloidal chitin. a. Moisten a cooled mortar with acetone and thoroughly mix ball milk chitin and concentrated hydrochloric acid. b Disperse dropwise into a large amount of cold water while stirring well. c Wash with water by centrifuging at 18000g for 10 minutes. d Process in a Waring blender for 10 minutes. e Centrifuge at 18000g for 10 minutes to collect colloidal chitin, and further wash with 0.025M Tris-HCl buffer (PH7.2). The colloidal chitin thus obtained is used after being dispersed in an appropriate buffer. Example 1 (1) Culture for enzyme production 1 part tap water, 15 g peptone, 5 parts yeast extract
g, add 0.68 g of potassium dihydrogen phosphate, 1N
- Pour 5 ml of the preculture medium adjusted to pH 7.0 with sodium hydroxide into test tubes with cotton plugs and sterilize according to the usual method. This is then followed by bacteria from the storage medium.
A loopful of A52 was inoculated and cultured with shaking at 28°C for 24 hours. For the main culture, 1 part of tap water, 10 g of shrimp shell chitin, 2 g of glucose, 5 g of peptone, 10 g of yeast extract, 0.68 g of potassium dihydrogen phosphate,
Add 3 g of sodium chloride and adjust the pH to 7.0 with 1N sodium hydroxide, then add 500 ml of main culture medium.
Dispense 70 ml into volumetric flasks and sterilize according to standard methods. Next, 1 ml of the preculture medium after completion of culture was inoculated into this, and cultured with shaking (240 rpm) at 28°C for 72 hours. (2) Preparation of crude enzyme solution After culturing, after removing the bacterial cells from the main culture medium by centrifugation at 18,000 g for 20 minutes, add sodium azide to a final concentration of 0.02% to prevent contamination with bacteria. It was added as a culture solution. Next, solid ammonium sulfate was gradually added to the culture solution to achieve 80% saturation while cooling to 0° C. and stirring gently with a stirrer to perform salting out. At this time, make sure that the pH of the culture solution does not lean toward the acidic side.
The pH was maintained around 7.0 with 1N sodium hydroxide. Salting out is carried out at 1°C while cooling to 0 to 4°C.
I went for more than an hour. The protein precipitate precipitated by salting out was collected by centrifugation at 18,000 g for 20 minutes. Add this precipitate to 0.1M in one-tenth volume of the culture solution.
Dissolved in Tris-HCl buffer (PH7.0), 0-
After cooling to 4 and standing for 1 hour, remove 18000g of insoluble matter.
The supernatant was removed by centrifugation for 20 minutes, and the supernatant was used as a crude enzyme solution and stored frozen at -20°C. (3) Separation of chitinase and chitobiase by chitin adsorption Based on the difference in affinity of both enzymes for colloidal chitin, they were separated as follows. 25mM containing 75ml of crude enzyme solution (approximately 887U of chitinase) and 2.5g of colloidal chitin (dry weight)
Mix 300ml of Tris-HCl buffer (PH7.2),
Chitinase was adsorbed onto colloidal chitin for 1 hour under ice cooling with occasional stirring. then 18000
Centrifugation was performed for 20 minutes at 100 g for 20 minutes, and the supernatant was used as the chitobiase fraction. (4) Purification of chitobiase The chitobinase fraction obtained in (3) of Example 1 was concentrated in an evaporator in the presence of 1mM PMSF, and then dialyzed overnight against 5mM Tris-HCl buffer (PH7.2). The product was ion-exchanged on a CM-cellulose column (2.6 x 45 cm) equilibrated with 50 mM acetate buffer (PH5.2). The elution was performed using a sodium chloride concentration gradient from 0 to 0.3M, and the flow rate was 40 ml/hour. Next, the eluate was concentrated and desalted by ultrafiltration, and then added to a 25mM Tris-HCl buffer (PH7.2) containing 0.05M sodium chloride.
DEAE equilibrated with SEFAEDEX A-50
column chromatogram (column size
2.6×45 cm) Purified chitobiase was obtained. In addition,
Elution was with a sodium chloride concentration gradient of 0.05→0.2M.
The flow rate was 44 ml/hour. The purification degree and recovery rate of tokibiase in each of the above operation steps are shown in Table 1, and the column chromatogram using DEAE-Sephadex A-50 is shown in Table 1.
As shown in the figure. As is clear from Table 1, the chitobiase in the crude enzyme solution was purified to 144 medium by the above procedure.
第１図はCM−セルロースのカラムによりイオ
ン交換して得られたキトビアーゼ溶出液のDEAE
−セフアデツクスＡ−50によるカラムクロマトグ
ラムを示す図面である。
Figure 1 shows the DEAE of chitobiase eluate obtained by ion exchange with a CM-cellulose column.
- It is a drawing showing a column chromatogram using Sephadex A-50.
Claims (1)
スA25（微工研菌寄第7206号）を培地に培養し、
その培養物から下記の理化学的性質を有するキト
ビアーゼを採取することを特徴とするキトビアー
ゼの製造法。 作用：キチナーゼと共に作用してキチンを分
解する。 至適PH：PH7.0 Km：ｐ−ニトロフエニル−Ｎ−アセチルグ
ルコサミニド0.17ｍＭ PH安定性：37℃で30分処理した場合、PH6.0
〜PH9.0において90％以上の残存活性を示す。 至適温度：PH7.0において、ｐ−ニトロフエ
ニル−Ｎ−アセチルグルコサミニドを基質とし
た場合50℃付近にある。 温度安定性：ｐ−ニトロフエニル−Ｎ−アセ
チルグルコサミニド基質で、PH7.0において、
０〜40℃、30分処理で80％以上の残存活性を示
す。 等電点：PH6.8付近 分子量：115000[Scope of Claims] 1. Cultivating Aeromonas hydrophila subanerogenes A25 (Feikoken Bacteria No. 7206) in a medium,
A method for producing chitobiase, which comprises collecting chitobiase having the following physicochemical properties from the culture. Action: Works together with chitinase to degrade chitin. Optimal PH: PH7.0 Km: p-nitrophenyl-N-acetylglucosaminide 0.17mM PH stability: PH6.0 when treated at 37℃ for 30 minutes
Shows residual activity of 90% or more at ~PH9.0. Optimum temperature: At pH 7.0, when p-nitrophenyl-N-acetylglucosaminide is used as a substrate, it is around 50°C. Temperature stability: p-nitrophenyl-N-acetylglucosaminide substrate at pH 7.0;
Shows residual activity of 80% or more after treatment at 0-40°C for 30 minutes. Isoelectric point: around PH6.8 Molecular weight: 115000
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16296083A JPS6054682A (en) | 1983-09-05 | 1983-09-05 | Chitobiase and its production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16296083A JPS6054682A (en) | 1983-09-05 | 1983-09-05 | Chitobiase and its production |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6054682A JPS6054682A (en) | 1985-03-29 |
| JPH0257917B2 true JPH0257917B2 (en) | 1990-12-06 |
Family
ID=15764547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16296083A Granted JPS6054682A (en) | 1983-09-05 | 1983-09-05 | Chitobiase and its production |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6054682A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56140406A (en) * | 1980-04-03 | 1981-11-02 | Kubota Ltd | Automatic control device of agricultural machine |
-
1983
- 1983-09-05 JP JP16296083A patent/JPS6054682A/en active Granted
Non-Patent Citations (1)
| Title |
|---|
| JOURNAL OF CLINICAL MICRODIOLOGY=1982 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6054682A (en) | 1985-03-29 |
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