JPH0343080A - Production of novel transglutaminase derived from streptoverticillium - Google Patents
Production of novel transglutaminase derived from streptoverticilliumInfo
- Publication number
- JPH0343080A JPH0343080A JP2147881A JP14788190A JPH0343080A JP H0343080 A JPH0343080 A JP H0343080A JP 2147881 A JP2147881 A JP 2147881A JP 14788190 A JP14788190 A JP 14788190A JP H0343080 A JPH0343080 A JP H0343080A
- Authority
- JP
- Japan
- Prior art keywords
- streptoverticillium
- transglutaminase
- culture
- activity
- btg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108060008539 Transglutaminase Proteins 0.000 title claims abstract description 28
- 102000003601 transglutaminase Human genes 0.000 title claims abstract description 28
- 241000187747 Streptomyces Species 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 125000002252 acyl group Chemical group 0.000 claims abstract description 6
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 5
- 238000006276 transfer reaction Methods 0.000 claims abstract description 5
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 238000000746 purification Methods 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract description 3
- 102000009127 Glutaminase Human genes 0.000 abstract description 2
- 108010073324 Glutaminase Proteins 0.000 abstract description 2
- 235000013305 food Nutrition 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 2
- 230000000050 nutritive effect Effects 0.000 abstract 2
- 241000499056 Streptomyces griseocarneus Species 0.000 abstract 1
- 230000001419 dependent effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 30
- 102000004190 Enzymes Human genes 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 18
- 239000000243 solution Substances 0.000 description 14
- 239000000758 substrate Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 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
- 150000003839 salts Chemical class 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000520730 Streptomyces cinnamoneus Species 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- SOUXAAOTONMPRY-NSHDSACASA-N 2-[[(2s)-5-amino-5-oxo-2-(phenylmethoxycarbonylamino)pentanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)N)NC(=O)OCC1=CC=CC=C1 SOUXAAOTONMPRY-NSHDSACASA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 239000002518 antifoaming agent Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- JIMLDJNLXLMGLX-JTQLQIEISA-N (2s)-5-amino-5-oxo-2-(phenylmethoxycarbonylamino)pentanoic acid Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 JIMLDJNLXLMGLX-JTQLQIEISA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- AIVHZBIRKPAQGR-JTQLQIEISA-N 2-[[(2s)-4-amino-4-oxo-2-(phenylmethoxycarbonylamino)butanoyl]amino]acetic acid Chemical group OC(=O)CNC(=O)[C@H](CC(=O)N)NC(=O)OCC1=CC=CC=C1 AIVHZBIRKPAQGR-JTQLQIEISA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- JEFZIKRIDLHOIF-BYPYZUCNSA-N Gln-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(O)=O JEFZIKRIDLHOIF-BYPYZUCNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101000933607 Homo sapiens Protein BTG3 Proteins 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102100026035 Protein BTG3 Human genes 0.000 description 1
- 101710123874 Protein-glutamine gamma-glutamyltransferase Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 101100438245 Solanum tuberosum PCM8 gene Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum 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
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids 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
- 239000012736 aqueous medium Substances 0.000 description 1
- 125000001942 asparaginyl group Chemical group 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 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 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 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
- 235000013351 cheese Nutrition 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- SWXVUIWOUIDPGS-UHFFFAOYSA-N diacetone alcohol Natural products CC(=O)CC(C)(C)O SWXVUIWOUIDPGS-UHFFFAOYSA-N 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 108010032995 epsilon-(gamma-glutamyl)-lysine Proteins 0.000 description 1
- JPKNLFVGUZRHOB-YUMQZZPRSA-N epsilon-(gamma-glutamyl)lysine Chemical compound OC(=O)[C@@H](N)CCCCNC(=O)CC[C@H](N)C(O)=O JPKNLFVGUZRHOB-YUMQZZPRSA-N 0.000 description 1
- -1 etc. Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000013618 yogurt Nutrition 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
Abstract
Description
【発明の詳細な説明】
［利用９腎１
本発明は、ストレプトベルチシリウム属由来の新規トラ
ンスグルタミナーゼの製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Utilization 9 Kidney 1] The present invention relates to a method for producing a novel transglutaminase derived from the genus Streptoverticillium.
更に詳しくは、新規トランスグルタミナーゼ生産能を有
するストレプトベルチシリウム属に属する菌株を栄養培
地に培養し、該培養物より新規トランスグルタミナーゼ
を採取することを特徴とするストレプトベルチシリウム
属由来の新規トランスグルタミナーゼの製造法に関する
。More specifically, a new transglutaminase derived from the genus Streptoverticillium is produced by culturing a strain belonging to the genus Streptoverticillium having the ability to produce a novel transglutaminase in a nutrient medium, and collecting the novel transglutaminase from the culture. This invention relates to a method for producing glutaminase.
トランスグルタミナーゼは、ペプチド鎖内にあるグルタ
ミン残基のγ−カルボキシアミド基のアシル転移反応を
触媒する酵素である。Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxyamide group of a glutamine residue within a peptide chain.
このトランスグルタミナーゼは、アシル受容体としてタ
ンパク質中のりジン残基のε−アミノ基が作用すると、
分子内及び分子間にε−（γ−Ｇｌｕ）−Ｌｙｓ架橋結
合が形成される。また水がアシル受容体として機能する
ときは、グルタミン残基が脱アミド化されグルタミン酸
残基になる反応を進行させる酵素である。This transglutaminase is activated when the ε-amino group of the lysine residue in the protein acts as an acyl acceptor.
ε-(γ-Glu)-Lys crosslinks are formed within and between molecules. Also, when water functions as an acyl acceptor, it is an enzyme that proceeds with the reaction of deamidating glutamine residues to become glutamic acid residues.
また、本発明の新規トランスグルタミナーゼを利用して
製造されるタンパク質のゲル化物は、従来のゲル状食品
、ゲル状化粧料をはじめとしてヨーグルト、ゼリー、チ
ーズなどとして用いられる。Furthermore, the protein gel produced using the novel transglutaminase of the present invention can be used in conventional gel foods, gel cosmetics, yogurt, jelly, cheese, and the like.
［従来の技術］
トランスグルタくナーゼはこれまで動物由来のものが知
、られている。例えばモルモ゛ットの肝臓（Ｃｏｎｎｅ
ｌｌａｎ ｅｔ ａｌ、、ジャーナル・オブ・バイオロ
ジカル・ケミストリー（Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏ
ｌＯｇｉｃａｌＣｈｅ１ｉＳｔｒ’／）２４６巻 ４号
１０９３〜１０９８頁（１９７１））及び哺乳動物の
臓器、血液に広く分布しくＦｏｌｋ ｅｔａｌ、、アト
パンセス・イン・エンザイモロジ−（Ａｄｖａｎｃｅｓ
ｉｎ ＥｎｚｙＩｌｏｌｏｇｙ）３８巻 １０９〜１
９１ 頁（１９７３）、Ｆｏｌｋ ｅｔ ａｌ、、アト
パンセス・イン・プロティン・ケミストリー（ＡｄＶａ
ｎＣｅＳ ｉｎ ＰｒｏｔｅｉｎＣｈｅＩＩｉｓｔｒｙ
） ３１巻１〜１３３頁（１９７７）） 、その酵素の
特徴も研究されている。[Prior Art] Transglutanases derived from animals have been known so far. For example, guinea pig liver (Conne
llan et al., Journal of Biological Chemistry
Vol. 246, No. 4, pp. 1093-1098 (1971)) and widely distributed in mammalian organs and blood.
in EnzyIlology) Volume 38 109-1
91 (1973), Folk et al., Atopanthes in Protein Chemistry (AdVa
nCeS in ProteinCheIIIstry
), Vol. 31, pp. 1-133 (1977)), and the characteristics of the enzyme have also been studied.
しかし、現時点ではストレプトベルチシリウム属由来の
トランスグルタミナーゼについては報告されていない。However, at present, transglutaminase derived from the genus Streptoverticillium has not been reported.
［発明が解決しようとする問題点］
従来トランスグルタミナーゼの供給は動物に由来してい
るため実用性を考慮した場合、供給量、供給費用、保存
費用、ｖＩ製の困難さ等の種々の面から不利でありこの
ままでは産業上の利用への可能性はほとんど考えられな
かった。[Problems to be solved by the invention] Conventionally, the supply of transglutaminase is derived from animals, so when considering practicality, there are various problems such as supply amount, supply cost, storage cost, and difficulty in producing VI. It was disadvantageous, and if it remained as it was, there was almost no possibility of industrial use.
従って、本発明の課題は供給量、コストの面、精製の容
易さ等のいずれの面からも問題はなく、しかも反応にＣ
ａ２＋を必要としなくともよい点等、実用性の高いスト
レプトベルチシリウム属由来の新規トランスグルタミナ
ーゼの製造法の提供である。Therefore, the problem of the present invention is that there is no problem in terms of supply amount, cost, ease of purification, etc.
The present invention provides a method for producing a novel transglutaminase derived from the genus Streptoverticillium, which is highly practical in that it does not require a2+.
Ｅ問題点を解決するための手段］
これまで動物由来の酵素が検討されてきたが実用性に欠
けるため、本発明者等は給源を微生物に求め広く検索を
行った結果、ストレプトベルチシリウム属の菌について
Ｃａ２＋非存在下でもペプチド鎖内のグルタミン残基の
γ−カルボキシアミド基のアシル転移反応を触媒する従
来にない新規トランスグルタミナーゼ産生能があること
を見い出し、本発明を完成するに至った。すなわち、本
発明はＣａ２＋非依存性の、ペプチド鎖内のグルタミン
残基のγ−カルボキシアミド基のアシル転移反応を触媒
する新規トランスグルタミナーゼ生産能を有するストレ
プトベルチシリウム属に属する菌株を栄養培地に培養し
、該培養物より該新規トランスグルタミナーゼを採取す
ることを特徴とするストレプトベルチシリウム属由来の
新規トランスグルタミナーゼの製造法に関する。[Means for Solving Problem E] Enzymes derived from animals have been studied so far, but they lack practicality, so the present inventors conducted a wide search for the source in microorganisms, and found that Streptoverticillium spp. The present inventors have discovered that this bacterium has the ability to produce an unprecedented novel transglutaminase that catalyzes the acyl transfer reaction of the γ-carboxyamide group of the glutamine residue in the peptide chain even in the absence of Ca2+, and has completed the present invention. . That is, the present invention provides a nutrient medium using a strain belonging to the genus Streptoverticillium that has the ability to produce a novel transglutaminase that catalyzes a Ca2+-independent transacyl transfer reaction of the γ-carboxyamide group of a glutamine residue in a peptide chain. The present invention relates to a method for producing a novel transglutaminase derived from the genus Streptoverticillium, which comprises culturing and collecting the novel transglutaminase from the culture.
ストレプトベルチシリウム属の菌を具体的に示すと、ス
トレプトベルチシリウム、・グリセオカルネウム（Ｓｔ
ｒｅｌ）ｔＯＶｅｒｔｉｃｉｌｌｊｕｌｌ （ｌｒｉｓ
ｅＯｃａｒｒｌｅｕｌｌ）ＩＦＯ１２７７６、ストレプ
トベルチシリウム・シナモネウム・サブ・エスピー・シ
ナモネウム
（ＳｔｒｅｐｔｏｖｅｒｔｉｃｉｌｌｉｕｌＩｃｉｎｎ
ａｍｏｎｅｕｎ ｓｕｂ ｓｐ。Specific examples of bacteria belonging to the genus Streptoverticillium include Streptoverticillium, Griseocarneum (St
rel)tOVerticilljull (lris
eOcarrleull) IFO12776, Streptoverticillium cinnamoneum subsp.
amoneun sub sp.
ｃｉｎｎａｍｏｎｅｕｍ）ＩＦＯ１２８５２，ストレプ
トベルチシリウム・モバラエンス（Ｓｔｒｅｐｔｏｖｅ
ｒｔｉＣｉｌｌｉｕｌｌｎｏｂａｒａｅｎｓｅ）ＩＦｏ
１３８１９等があげられる。cinnamoneum) IFO12852, Streptoverticillium mobaraens (Strepttove
rtiCilliullnobaraense)IFo
Examples include 13819.
これら微生物を培養し、ストレプトベルチシリウム属由
来のトランスグルタミナーゼ（尚、以後ＢＴＧａｓｅと
記す）を取得するための培養法及び精製法等について述
べる。A culture method, a purification method, etc. for culturing these microorganisms and obtaining transglutaminase (hereinafter referred to as BTGase) derived from the genus Streptoverticillium will be described.
本発明を実施するにあたり、その培養形態としては液体
培養、固体培養いずれも可能であるが、工業的には深部
通気撹拌培養を行うのが有利である。In carrying out the present invention, both liquid culture and solid culture are possible, but from an industrial perspective it is advantageous to perform deep aeration agitation culture.
又、使用する栄！！培地の培養源としては一般に微生物
培養に用いられる炭素源、窒素源、無機塩及びその他の
微量栄ｉ源の他、ストレブトベルチシリウム属に属する
微生物の利用出来る栄養源であれば全て使用出来る。培
地の炭素源としてはブドウ糖、シヨ糖、可溶性デンプン
「ラスターゲン」〈商品名９日電化学社製）、グリセリ
ン、デキストリン、Ｒ粉等の他、脂肪酸、油脂、有機酸
などが単独で又は組合せて用いられる。窒素源としては
無機窒素源、有機窒素源のいずれも使用可能であり、無
機栄養源としては硝酸アンモニウム、＠酸アンモニウム
、尿素、硝酸ソーダ、ＦＡ化アンモニウム等が挙げられ
る。又有機窒素源としては大豆、米、トウモロコシ、小
麦などの粉、糠、脱脂粕をはじめコーンステイープリカ
ー、ペプトン。Also, Sakae to use! ! As a culture source for the medium, in addition to carbon sources, nitrogen sources, inorganic salts, and other trace nutrient sources that are generally used for microbial culture, any nutrient source that can be used by microorganisms belonging to the genus Strebtoverticillium can be used. . Carbon sources for the culture medium include glucose, sucrose, soluble starch "Lastargen" (trade name: manufactured by 9th Denkagaku Co., Ltd.), glycerin, dextrin, R powder, etc., as well as fatty acids, fats and oils, organic acids, etc., singly or in combination. used. As the nitrogen source, either an inorganic nitrogen source or an organic nitrogen source can be used, and examples of the inorganic nutrient source include ammonium nitrate, ammonium acid, urea, sodium nitrate, and ammonium FA. Organic nitrogen sources include soybean, rice, corn, wheat flour, bran, defatted lees, cornstarch liquor, and peptone.
肉エキス、カゼイン、アミノ酸、酵母エキス等が挙げら
れる。無機塩及び微量栄養素としてはリン酸、マグネシ
ウム、カリウム、鉄、カルシウム。Examples include meat extract, casein, amino acids, yeast extract, etc. Inorganic salts and micronutrients include phosphoric acid, magnesium, potassium, iron, and calcium.
亜鉛等の塩類の他ビタミン、非イオン界面活性剤。Salts such as zinc, vitamins, and nonionic surfactants.
消泡剤等の菌の生育やＢＴＧａｓｅの生産を促進するも
のであれば必要に応じて使用出来る。Any antifoaming agent that promotes bacterial growth and BTGase production can be used as needed.
培養は好気的条件で、培養温度は菌が発育しＢＴＧａｓ
ｅが産生する範囲であれば良く、好ましくは２５〜３５
℃である。培養時間は条件により異なるがＢＴＧａｓｅ
Ｓ最も産生される時間まで培養すれば良く、通常２〜４
日程度である。The culture is carried out under aerobic conditions, and the culture temperature is set to allow the bacteria to grow.
It may be within a range that produces e, preferably 25 to 35
It is ℃. Although the culture time varies depending on the conditions, BTGase
It is sufficient to culture until the time when S is produced the most, usually 2 to 4
It is about 1 day.
ＢＴＧａｓｅは液体培養では培養液中に溶解されており
、培養終了後培養液より固形分を除いた培養ろ液より採
取される。培養ろ液より ＢＴＧａｓｅを精製するには
通常酵素精製に用いられるあらゆる方法が使用出来る。In liquid culture, BTGase is dissolved in the culture solution, and after the completion of culture, it is collected from the culture filtrate after removing the solid content from the culture solution. To purify BTGase from culture filtrate, any method commonly used for enzyme purification can be used.
例えばエタノール、アセトン、イソプロピルアルコール
等の有機溶媒による処理、硫安１食塩等による塩析、透
析、限外ろ適法、イオン交換クロマトグラフィー、吸着
クロマトグラフィー、ゲルろ過、吸着剤、等電点分画等
の方法が使用出来る。For example, treatment with organic solvents such as ethanol, acetone, and isopropyl alcohol, salting out with ammonium sulfate, dialysis, ultrafiltration, ion exchange chromatography, adsorption chromatography, gel filtration, adsorbents, isoelectric point fractionation, etc. method can be used.
又これらの方法を適当に組合せることによりＢＴＧａｓ
ｅの精製度が上る場合は適宜組合せて行うことが出来る
。Also, by appropriately combining these methods, BTGas
If the degree of purification of e is increased, it can be carried out in combination as appropriate.
こうしてこれらの方法によって得られた酵素液に安定化
剤として各種の塩類、糖類、蛋白質、脂質、界面活性剤
等を加えあるいは加えることなく、限外ろ過濃縮、逆浸
透濃縮、減圧乾燥、凍結乾燥。The enzyme solution obtained by these methods can be subjected to ultrafiltration concentration, reverse osmosis concentration, vacuum drying, and freeze drying, with or without adding various salts, sugars, proteins, lipids, surfactants, etc. as stabilizers. .
噴霧乾燥の方法を施すことにより液状又は固形のＩ製Ｂ
ＴＧａｓｅを得ることが出来る。Liquid or solid I made B by applying the spray drying method.
TGase can be obtained.
ＢＴＧａｓｅの活性測定はベンジルオキシカルボニル−
し−グルタミニルグリシンとヒトＯキシルアミンを基質
としてＣａ２＋非存在下で反応を行い、生成したヒドロ
キサム酸をトリクロロ酢酸存在下で銖錯体を形成させ５
２５ｎｉの＠収を測定し、ヒドロキサム酸の量を検量線
より求め活性を算出する。BTGase activity measurement is based on benzyloxycarbonyl-
A reaction is carried out in the absence of Ca2+ using glutaminylglycine and human O-xylamine as substrates, and the resulting hydroxamic acid is formed into a complex in the presence of trichloroacetic acid.5
The yield of 25ni is measured, and the amount of hydroxamic acid is determined from the calibration curve to calculate the activity.
８ＴＧａｓｅ活性は特に記載しないかぎり以下に記載す
る方法により測定した。8TGase activity was measured by the method described below unless otherwise specified.
〈活性測定法〉
試薬Ａ ０．２Ｈトリスｊ３ｉｉＭ！緩ｉｌｉ液（ｐＨ
６，０）０．１Ｈヒドロキシルアミン
ｏ、ｏｉｓ還元型グルタチオン
０、０３８ベンジルオキシカルボニル−試薬８ ３Ｎ
塩酸
１２％トリクロロ酢酸
５％ＦｅＣｌ −６目，０
（０．ＩＮＨＣＩに溶解）
上記溶液の１：１：１の混合液を試薬Ｂとする。<Activity measurement method> Reagent A 0.2H Tris j3iiM! Mild lily solution (pH
6,0) 0.1H hydroxylamine o, ois reduced glutathione 0,038 benzyloxycarbonyl-reagent 8 3N
Hydrochloric acid 12% Trichloroacetic acid 5% FeCl -6,0 (dissolved in 0.INHCI) A 1:1:1 mixture of the above solutions was used as Reagent B.
酵素液の０． ０５ｄに試薬Ａ０．５−を加えて混合し
３７゛Ｃで１０分間反応後、試薬Ｂ０．５−を加えて反
応停止とＦｅ錯体の形成を行った後５２５−ｒ＋ｍの吸
光度を測定する。対照としてあらかじめ熱失活させた酵
素液を用いて同様に反応させたものの吸光度を測定し、
酵素液との吸光度差を求める。別に酵素液のかわりにし
一グルタミン酸γ−モノヒドロキサム酸を用いて検量線
を作成し、前記吸光度差により生成されたヒドロキサム
酸の量を求め、１分間に１μモルのヒドロキサム酸を生
成する酵素活性を１単位とした。0 of the enzyme solution. Add reagent A0.5- to 05d, mix and react at 37°C for 10 minutes, then add reagent B0.5- to stop the reaction and form an Fe complex, and then measure the absorbance of 525-r+m. As a control, we performed a similar reaction using an enzyme solution that had been heat-inactivated in advance, and measured the absorbance.
Determine the absorbance difference with the enzyme solution. Separately, a calibration curve was created using monoglutamic acid γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined by the difference in absorbance, and the enzyme activity to produce 1 μmol of hydroxamic acid per minute was determined. It was set as 1 unit.
このようにして得られる精製ＢＴＧａｓｅの酵素化学的
性質を以下に述べる。尚、ストレプトベルチシリウム馬
肉の菌株の種類により
ＢＴＧａＳｅの酵素化学的性質について若干の相違点が
みられるので、それぞれの菌株の生産するＢＴＧａｓｅ
、即ちストレプトベルチシリウム・モバラエンス（Ｓｔ
ｒｅｐｔｏｖｅｒｔｉｃｉｌｌｉｕｍ ｎｏｂａｒａｅ
ｎｓｅ）ＩＦｏ １３８１９のトランスグルタミナーゼ
（ＢＴＧ−１と命名〉、ストレプトベルチシリウム・グ
リセオ力ルネウムｔｓｔｒｅｐｔｏｖｅｒｔ ｉｃ ｉ
ｌ Ｉ ｉｕｌｌｏｒｉｓｅｏｃａｒｎｅｕｍ） Ｉ
ＦＯ１２７７６のトランスグルタミナーゼ（ＢＴＧ−２
と命名）、ストレプトベルチシリウム・シナモネウム・
サブ・エスピー・シナモネウム（Ｓｔｒｅｐｔｏｖｅｒ
ｔｉｃｉｌｌｉｕｍ ｃｉｎｎａｍｏｎｅｕｎｓｕｂ
ｓｐ、ｃｉｎｎａｍｏｎｅｕｍ）） ＩＦＯ１２８５２
のトランスグルタミナーゼ（ＢＴＧ−３と命名）につい
ての酵素化学的性質を記載するとともに、それらを包含
したものをＢＴＧａｓｅの酵素化学的性質とする。The enzymatic chemical properties of the purified BTGase thus obtained will be described below. In addition, there are some differences in the enzymatic chemical properties of BTGaSe depending on the type of Streptoverticillium horsemeat strain, so the BTGase produced by each strain
, namely Streptoverticillium mobaraens (St
reptoverticillium nobarae
transglutaminase of IFo 13819 (named BTG-1), Streptoverticillium griseolneum tstreptovertic i
I
FO12776 transglutaminase (BTG-2
), Streptoverticillium cinnamoneum
Subsp. Cinnamonium (Streptover)
ticillium cinnamone unsub
sp,cinnamoneum)) IFO12852
The enzymatic chemical properties of the transglutaminase (named BTG-3) will be described, and the enzymatic chemical properties of BTGase will include these.
ａ）至適ＤＨ：６〜７
基質としてベンジルオキシカルボニル−Ｌ−グルタミニ
ルグリシンとヒドロキシルアくンを使用し、３７℃、１
０分反応で作用至適ｐＨ範囲を求めた。尚、ＢＴＧ−１
の至適１）Ｈは６〜７にあり、ＢＴＧ−２の至適ｐＨは
６〜７付近にあり、ＢＴＧ−３の至適ｐＨは６〜７付近
にある（第１図、第５図及び第９図参照）。a) Optimum DH: 6-7 Using benzyloxycarbonyl-L-glutaminylglycine and hydroxyluacan as substrates, at 37°C, 1
The optimum pH range for action was determined in a 0 minute reaction. Furthermore, BTG-1
The optimum pH of 1) H is in the range of 6 to 7, the optimum pH of BTG-2 is in the vicinity of 6 to 7, and the optimum pH of BTG-3 is in the vicinity of 6 to 7 (Fig. 1, Fig. 5). and Figure 9).
ｂ）至適温度：４５〜５５℃
基質としてベンジルオキシカルボニル−Ｌ−グルタミニ
ルグリシンとヒドロキシルアミンを使用し、ｐＨ６，１
０分反応での作用至適温度範囲を求めた。尚、ＢＴＧ−
１の至適温度は５５℃付近であり、８ＴＧ−２の至適温
度は４５℃付近であり、ＢＴＧ−３の至適温度は４５℃
付近にある（第２図、第６図及び第１０図参照）。b) Optimal temperature: 45-55°C Using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates, pH 6.1
The optimum temperature range for 0 minute reaction was determined. Furthermore, BTG-
The optimal temperature for 1 is around 55°C, the optimal temperature for 8TG-2 is around 45°C, and the optimal temperature for BTG-3 is 45°C.
(See Figures 2, 6 and 10).
ｃ ） ｐＨ安定性：ｐＨ５〜９
３７℃、１０分間処理でのｐＨ安定性を求めた。尚、Ｂ
ＴＧ−１はｐＨ５〜９で安定であり、ＢＴＧ−２はｐＨ
は５〜９で安定であり、ＢＴＧ−３はＥＩＨ６〜９で安
定である（第３図、第７図及び第１１図参照）。c) pH stability: pH 5 to 9 The pH stability was determined by treatment at 37°C for 10 minutes. Furthermore, B
TG-1 is stable at pH 5-9, and BTG-2 is stable at pH 5-9.
is stable at EIH of 5 to 9, and BTG-3 is stable at EIH of 6 to 9 (see Figures 3, 7, and 11).
ｄ）温度安定性 ｐＨ７で１０分間処理での温度安定範囲を求めた。d) Temperature stability The temperature stability range after treatment at pH 7 for 10 minutes was determined.
４０℃では８０％以上、５０℃では５０〜８０％の活性
がそれぞれ残存した。尚、ＢＴＧ−１は４０℃では８８
％活性が残存し、５０℃では７４％活性が残存し、ＢＴ
Ｇ−２は４０℃では８６％活性が残存し、５０℃では５
６％活性が残存し、ＢＴＧ＝−，３は４０℃で８０％活
性が残存し、５０℃では５３％活性が残存する〈第４図
、第８図及び第１２図参照〉。More than 80% of the activity remained at 40°C, and 50-80% of the activity remained at 50°C. In addition, BTG-1 has a temperature of 88 at 40°C.
% activity remains, 74% activity remains at 50°C, BT
G-2 has 86% activity remaining at 40°C, and 5% activity remains at 50°C.
6% activity remains, BTG=-,3 has 80% activity remaining at 40°C and 53% activity remaining at 50°C (see Figures 4, 8 and 12).
ｅ）基質特異性
ＢＴＧａｓｅの各種合成基質とヒドロキシルアくンとの
反応を調べた。e) Substrate specificity The reaction between various synthetic substrates of BTGase and hydroxylua was investigated.
合成基質がベンジルオキシカルボニルアスパラギニルグ
リシン、ベンジルオキシカルボニルグルタミン、グリシ
ルグルタミニルグリシンの場合反応しない。No reaction occurs when the synthetic substrate is benzyloxycarbonylasparaginylglycine, benzyloxycarbonylglutamine, or glycylglutaminylglycine.
しかし、合成基質がペンジルオキシ力ルポニルグルタミ
ニルグリシンの場合反応性は最も高い。この時の各種合
成基質濃度は５１１Ｎとした。However, the reactivity is highest when the synthetic substrate is penzyloxyluponylglutaminylglycine. The concentration of various synthetic substrates at this time was 511N.
結果は表−１に示される。尚、表−１中のＣＢＩはベン
ジルオキシカルボニル基の略であり、ａ＋ｎはグルタミ
ル基の略であり、ＧＩｙはグリシル基の略であり、
Ａｓｎ
はアスパラギニル基の略
である。The results are shown in Table-1. In Table 1, CBI is an abbreviation for benzyloxycarbonyl group, a+n is an abbreviation for glutamyl group, GIy is an abbreviation for glycyl group, and Asn is an abbreviation for asparaginyl group.
表
ｆ）金属イオンの影響
活性測定系に１１Ｎ濃度になるように各種金属イオンを
加えて影響を調べた。Table f) Effect of metal ions Various metal ions were added to the activity measurement system to give a concentration of 11N, and the effects were investigated.
結果は表−２に示される。ＢＴ（３ａｓｅはＣｕ、Ｚｎ
”＋により活性が阻害される。The results are shown in Table-2. BT (3ase is Cu, Zn
``+ inhibits activity.
２十
表
ｈ〉阻害剤の影響
各阻害剤を１ｉＨになるように加え、２５℃、３０分放
置後、活性を測定した。Table 20 h〉Effect of inhibitors Each inhibitor was added at a concentration of 1iH, and after standing at 25°C for 30 minutes, the activity was measured.
結果は表−３に示される。８ＴＧａｓｅはパラクロロマ
ーキュリ−安息香酸（ＰＣＭ８と略する）、Ｎ−エチル
マレイミド（ＮＥＭと略する）、モノヨード酢酸により
活性が阻害される。The results are shown in Table-3. The activity of 8TGase is inhibited by parachloromercury-benzoic acid (abbreviated as PCM8), N-ethylmaleimide (abbreviated as NEM), and monoiodoacetic acid.
表 −３
表−３中ＰＭＳＦはフェニルメチルスルホニルフロオラ
イドの略である。Table 3 PMSF in Table 3 is an abbreviation for phenylmethylsulfonyl fluoride.
ｉ〉等電点：８．９〜９．９ アンホライン等電点電気泳動により求めた。i〉Isoelectric point: 8.9-9.9 It was determined by ampholine isoelectric focusing.
尚、ＢＴＧ−１の等電点（ｐＩ）は９付近であり、ＢＴ
Ｇ−２の等電点（ｐｌ）は９．７付近であり、ＢＴＧ−
３の等電点（ｐｌ）は９．８付近である。In addition, the isoelectric point (pI) of BTG-1 is around 9, and BT
The isoelectric point (pl) of G-2 is around 9.7, and BTG-
The isoelectric point (pl) of No. 3 is around 9.8.
ｊ）分子ｍ：約３８，０００〜約４１，０００ＳＯＳデ
イスク電気泳動法より求めた。尚、ＢＴＧ−１の分子量
は約３８．０００であり、ＢＴＧ−２の分子量は約４１
，０００であり、８ＴＧ−３の分子量は約４１，０００
である。j) Molecule m: about 38,000 to about 41,000 determined by SOS disk electrophoresis. The molecular weight of BTG-1 is approximately 38,000, and the molecular weight of BTG-2 is approximately 41.
,000, and the molecular weight of 8TG-3 is approximately 41,000.
It is.
次に、ＢＴＧａｓｅとモルモット肝由来のトランスグル
タミナーゼ（以後ＭＴＧａｓｅと記す）との性質を比較
する。尚、ＭＴＧａｓ−ｅは特開昭５８−１４９６４５
号に記載された方法で調製した。Next, the properties of BTGase and transglutaminase derived from guinea pig liver (hereinafter referred to as MTGase) will be compared. Furthermore, MTGas-e is based on Japanese Patent Application Laid-Open No. 58-149645.
It was prepared by the method described in No.
表−４には各酵素化学的性質の比較を、表−５にはＣａ
２＋の活性に及ぼす影響を示す。表−４及び表−５より
明らかなように従来上として研究されているＭＴＧａｓ
ｅとストレプトベルチシリウム属由来のＢＴＧａｓｅと
では酵素化学的性質において種々の差が見られ、特に温
度安定性、分子量、等電点、基質特異性に差が見られる
。また、Ｃａ２＋の存在下及び非存在下のいずれにおい
ても本発明のトランスグルタミナーゼは作用する点等で
も明らかな差がみられる。従って、本発明のＢＴＧａｓ
ｅはＭＴＧａｓｅとはその性質を明らかに異にするもの
であり、新規トランスグルタミナーゼである。Table 4 shows a comparison of the chemical properties of each enzyme, and Table 5 shows Ca
The effect on 2+ activity is shown. As is clear from Tables 4 and 5, MTG
There are various differences in enzymatic chemical properties between BTGase derived from the genus Streptoverticillium and BTGase, particularly in temperature stability, molecular weight, isoelectric point, and substrate specificity. Further, there is a clear difference in that the transglutaminase of the present invention acts both in the presence and absence of Ca2+. Therefore, the BTGas of the present invention
e is a novel transglutaminase whose properties are clearly different from MTGase.
表
以下に本発明の８ＴＧａｓｅの製造法について実施例に
て具体的に説明する。The method for producing 8TGase of the present invention will be specifically explained in Examples below.
１亙旦ユ
ストレプトベルチシリウム・モバラエンス（Ｓｔｒｅｐ
ｔｏｖｅｒｔｉｃｉｌｌｉｕｉ １ｏｂａｒａｅｎｓｅ
）ＩＦＯ１３８１９を培地組成ポリペプトン０．２％、
グルコース０．５％。1 year ago, Justreptoverticillium mobalaens (Strep
toverticilliui 1obaraense
) IFO13819 medium composition polypeptone 0.2%,
Glucose 0.5%.
リン酸二カリウム０．２％、硫酸マグネシウム０．１％
からなる水性培地（ｐＨ７）２００ｄ Ｉｃ接種し３０
”Ｃ，４８時間培養し、得られた種培養液をポリペプト
ン２．０％、「ラスターゲン」０．２％、リン酸二カリ
ウム０．２％、硫酸マグネシウム０．１％、酵母エキス
０．２％、消泡剤としてポリオキシアルキレングリコー
ルの「アデカノール」 （商品名、旭電化社製品） Ｏ
，ＯＳ％からなる水性培地２０１ （ｐＨ７）に加え３
０℃で３日間培養後ろ過し、培養液１８．５１’ｌ！ｌ
た。Dipotassium phosphate 0.2%, magnesium sulfate 0.1%
An aqueous medium (pH 7) consisting of 200 d Ic inoculated with 30
``C, After culturing for 48 hours, the resulting seed culture solution was mixed with 2.0% polypeptone, 0.2% ``Lastargen'', 0.2% dipotassium phosphate, 0.1% magnesium sulfate, and 0.0% yeast extract. 2%, polyoxyalkylene glycol “Adekanol” (trade name, Asahi Denka product) as an antifoaming agent O
, OS% 201 (pH 7) plus 3
After culturing at 0°C for 3 days, strain the culture solution to 18.51'l! l
Ta.
このものの活性は０．３５ユニツト／ｄであった。The activity of this product was 0.35 units/d.
培養液を塩酸でｐＨ６，５に調製し、予め０．０５Ｍリ
ン酸緩衡液（ｐＨ６，５）で平衡化しておいたメタアク
リル酸系ポーラス型隣イオン交換樹脂の「アンバーライ
トＣＧ−５０４（商品名、ローム・アンド・ハース社製
品）のカラムに通した。この操作でトランスグルタ４ナ
ーゼは＠着された。さらに同緩衝液で不純蛋白質を洗い
流した後、更に０．０５〜０゜５Ｍの同緩衝液の濃度勾
配をつくり、通液して溶出液を分画回収し、比活性の高
い分画を集めた。The culture solution was adjusted to pH 6.5 with hydrochloric acid and equilibrated in advance with 0.05M phosphoric acid buffer (pH 6.5). (trade name, Rohm & Haas Co.) column. Transgluta-4nase was attached by this operation. After washing away impure proteins with the same buffer, further 0.05 to 0.5 M A concentration gradient of the same buffer solution was created, the eluate was collected in fractions, and the fractions with high specific activity were collected.
電導度を１０ｎｓ以下になるように稀釈′後「ブルーセ
ファロースＣＬ−６ＢＪ （商品名、ファルマシア・
ファインケミカル社製）のカラムに通した。After diluting the conductivity to 10 ns or less, "Blue Sepharose CL-6BJ (trade name, Pharmacia
The mixture was passed through a column manufactured by Fine Chemical Co., Ltd.).
この操作でトランスグルタミナーゼは吸着された。Transglutaminase was adsorbed by this operation.
更に０．０５Ｍリン酸緩衝液（ｐＨ７）で不純蛋白質を
洗い流した後、０〜１Ｍの食塩濃度勾配をつくり通液し
て溶出液を回収し比活性の高い画分を集めた。Furthermore, after washing away impure proteins with 0.05M phosphate buffer (pH 7), a 0-1M salt concentration gradient was created and the eluate was collected, and fractions with high specific activity were collected.
限外濾過膜のｒＡ Ｉ ＬｌｏｌｏＪ （商品名、旭
化或工業（株）製）を使い濃縮し、０．５Ｍの食塩を含
む０．０５Ｍリン酸緩衝液（ｐＨ７）を用いて平衡化さ
せた。It was concentrated using an ultrafiltration membrane rA I LloloJ (trade name, manufactured by Asahi Kaoru Kogyo Co., Ltd.) and equilibrated using 0.05M phosphate buffer (pH 7) containing 0.5M salt. .
得られたＩＩ縮液を同！ｌ樹液で予め平衡化しておいた
「セファデックスＧ−７５Ｊ （商品名、ファルマシ
ア・ファインケミカル社製）を含むカラムに通し、同緩
衝液を流して溶出液を分画した。The obtained II condensate is the same! The eluate was fractionated by passing the same buffer through a column containing Sephadex G-75J (trade name, manufactured by Pharmacia Fine Chemicals) that had been equilibrated with l sap.
この結果活性画分は単一のピークとして溶出された。こ
のものの比活性は培養ろ液に対し６２５倍であり、回収
率は４７％であった。As a result, the active fraction was eluted as a single peak. The specific activity of this product was 625 times that of the culture filtrate, and the recovery rate was 47%.
大１ｕ生２
実施例１と同様にしてストレプトベルチシリウム・グリ
セオ力ルネウム（Ｓｔｒｅｐｔｏｖｅｒｔ ｉｃ ｉ
ｌ ｌ ｉｕｍａｒｉｓｅｏｃａｒｎｅｕｌ） ＩＦＯ
１２７７６を３０℃で３日間培養後ろ過し培養液１９１
を得た。このものの活性は０゜２８Ｕ／ルであった。Streptoverticillium (Streptoverticillium) was grown in the same manner as in Example 1.
IFO
12776 was cultured at 30°C for 3 days and the culture solution 191 was filtered.
I got it. The activity of this product was 0.28 U/L.
実施例１と同様な方法で酵素を精製してＳＯＳディスク
電気泳動で単一の酵素を得た。The enzyme was purified in the same manner as in Example 1, and a single enzyme was obtained by SOS disk electrophoresis.
友簾旦ユ
実施例１と同様にしてストレプトベルチシリウム・シナ
モネウム・サブ・エスピー・シナモネウム（Ｓｔｒｅｐ
ｔｏｖｅｒｔｉｃｉｌｌｉｕｎ ｃｉｎｎａｎ＋ｏｎｅ
ｕｎ ｓｕｂ ｓｐ。In the same manner as in Example 1, Streptoverticillium cinnamoneum subsp. cinnamoneum (Strep
toverticilliun cinnan+one
un sub sp.
ｃｉｎｎａｌｏｎｅｕＩ） ＩＦｏ １２８５２を３０
℃で３日間培養後ろ過し、培養液１８．５ｊ！を得た。cinnaloneuI) IFo 12852 30
After culturing at ℃ for 3 days, strain the culture solution to 18.5j! I got it.
このものの酵素活性は０．５ｕ／−であった。The enzyme activity of this product was 0.5 u/-.
実施例１と同様な方法で酵素を精製してＳＤＳディスク
電気泳動で単一の酵素を得た。The enzyme was purified in the same manner as in Example 1, and a single enzyme was obtained by SDS disk electrophoresis.
【発明の効果］
本発明のストレプトベルチシリウム属由来のＢＴＧａｓ
ｅは安価に供給され、かつ精製も容易であるので実用性
が大である。[Effect of the invention] BTGas derived from the genus Streptoverticillium of the present invention
Since e is supplied at a low cost and can be easily purified, it has great practicality.
また、ＢＴＧａｓｅを用いることにより、カルシウム非
存在下でも酵素（ＢＴＧａｓｅ＞１度及び基質濃度が非
常に低いところで品質の優れたゲル化物を製造できると
いう利点がある。Furthermore, the use of BTGase has the advantage that a gelled product of excellent quality can be produced even in the absence of calcium, where the enzyme (BTGase>1 degree and the substrate concentration is very low).
第１図、第２図、第３図及び第４図は本願発明のＢＴＧ
−１の至適ｐＨ曲線、至適温度曲線、ｐ１１安定曲線及
び温度安定曲線を示すものであり、第５図、第６図、第
７図及び第８図は本願発明のＢＴＧ−２の至適ｐＨ曲線
、至適温度曲線、 ｐＨ安定曲線及び温度安定曲線を示
すものであり、第９図、第１０図、第１１図及び第１２
図は、本願発明のＢＴＧ３の至適ＯＨ曲線、至適温度曲
線、ｐ１１安定曲線及び温度安定曲線を示すものである
。
第
図
ｐＨ
第
図
０
０６０
温度（°Ｃ）
０
第
３
図
ｐＨ
第
図
温
度
（”Ｃ）
第
図
ｐＨ・
露
図
０
０６０
温度（”Ｃ）
０
露
図
ｐＨ
第
図
温
度 （°Ｃ）
第
図
ｐＨ
第
０
図
０
０
０
０
瀉
度
（”Ｃ）Figures 1, 2, 3, and 4 are BTGs of the present invention.
FIG. 5, FIG. 6, FIG. 7, and FIG. 8 show the optimum pH curve, optimum temperature curve, p11 stability curve, and temperature stability curve of BTG-1 of the present invention. This shows the optimum pH curve, optimum temperature curve, pH stability curve, and temperature stability curve, and Figs. 9, 10, 11, and 12.
The figure shows the optimal OH curve, optimal temperature curve, p11 stability curve, and temperature stability curve of BTG3 of the present invention. Figure pH Figure 0 060 Temperature (°C) 0 Figure 3 pH Figure Temperature (''C) Figure pH・Dew Figure 0 060 Temperature (''C) 0 Dew Figure pH Figure Temperature (°C) Figure pH 0 Figure 0 0 0 0 Temperature ("C)
Claims (1)
ミン残基のγ−カルボキシアミド基のアシル転移反応を
触媒する新規トランスグルタミナーゼ生産能を有するス
トレプトベルチシリウム属に属する菌株を栄養培地に培
養し、該培養物より該新規トランスグルタミナーゼを採
取することを特徴とするストレプトベルチシリウム属由
来の新規トランスグルタミナーゼの製造法。(1) Nutrients a strain belonging to the genus Streptoverticillium that has the ability to produce a novel transglutaminase that catalyzes a Ca^2^+-independent acyl transfer reaction of the γ-carboxyamide group of a glutamine residue in a peptide chain. A method for producing a novel transglutaminase derived from the genus Streptoverticillium, which comprises culturing it in a medium and collecting the novel transglutaminase from the culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2147881A JPH0343080A (en) | 1987-03-04 | 1990-06-06 | Production of novel transglutaminase derived from streptoverticillium |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4915787 | 1987-03-04 | ||
| JP62-49157 | 1987-03-04 | ||
| JP2147881A JPH0343080A (en) | 1987-03-04 | 1990-06-06 | Production of novel transglutaminase derived from streptoverticillium |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62165067A Division JPH0665280B2 (en) | 1987-03-04 | 1987-07-01 | Protein gelling agent and protein gelling method using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0343080A true JPH0343080A (en) | 1991-02-25 |
| JPH0523744B2 JPH0523744B2 (en) | 1993-04-05 |
Family
ID=26389516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2147881A Granted JPH0343080A (en) | 1987-03-04 | 1990-06-06 | Production of novel transglutaminase derived from streptoverticillium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0343080A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993020837A1 (en) * | 1992-04-21 | 1993-10-28 | Ajinomoto Co., Inc. | Remedy for wound |
| US6039901A (en) * | 1997-01-31 | 2000-03-21 | Givaudan Roure Flavors Corporation | Enzymatically protein encapsulating oil particles by complex coacervation |
| JP2016500266A (en) * | 2012-12-14 | 2016-01-12 | ヒルズ・ペット・ニュートリシャン・インコーポレーテッド | Method for preparing food composition |
| US11577670B2 (en) | 2018-11-29 | 2023-02-14 | Nishikawa Rubber Co., Ltd. | Weather strip, weather strip attachment structure, and weather strip attachment method |
-
1990
- 1990-06-06 JP JP2147881A patent/JPH0343080A/en active Granted
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993020837A1 (en) * | 1992-04-21 | 1993-10-28 | Ajinomoto Co., Inc. | Remedy for wound |
| US6039901A (en) * | 1997-01-31 | 2000-03-21 | Givaudan Roure Flavors Corporation | Enzymatically protein encapsulating oil particles by complex coacervation |
| US6325951B1 (en) | 1997-01-31 | 2001-12-04 | Givaudan Roure Flavors Corporation | Enzymatically protein-encapsulating oil particles by complex coacervation |
| JP2016500266A (en) * | 2012-12-14 | 2016-01-12 | ヒルズ・ペット・ニュートリシャン・インコーポレーテッド | Method for preparing food composition |
| US11577670B2 (en) | 2018-11-29 | 2023-02-14 | Nishikawa Rubber Co., Ltd. | Weather strip, weather strip attachment structure, and weather strip attachment method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0523744B2 (en) | 1993-04-05 |
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