JPH0476673B2 - - Google Patents
Info
- Publication number
- JPH0476673B2 JPH0476673B2 JP22822083A JP22822083A JPH0476673B2 JP H0476673 B2 JPH0476673 B2 JP H0476673B2 JP 22822083 A JP22822083 A JP 22822083A JP 22822083 A JP22822083 A JP 22822083A JP H0476673 B2 JPH0476673 B2 JP H0476673B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- reaction
- plp
- polymer
- activity
- 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
Links
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- 108090000790 Enzymes Proteins 0.000 claims description 65
- 229920001223 polyethylene glycol Polymers 0.000 claims description 18
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 10
- 229920001515 polyalkylene glycol Polymers 0.000 claims description 9
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 241000590020 Achromobacter Species 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 238000004132 cross linking Methods 0.000 claims description 4
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 claims description 4
- 230000000379 polymerizing effect Effects 0.000 claims description 3
- 108010053229 Lysyl endopeptidase Proteins 0.000 claims description 2
- WBGKWQHBNHJJPZ-LECWWXJVSA-N desonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O WBGKWQHBNHJJPZ-LECWWXJVSA-N 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 description 45
- 229920000642 polymer Polymers 0.000 description 31
- 230000000694 effects Effects 0.000 description 25
- 238000000034 method Methods 0.000 description 23
- 239000003960 organic solvent Substances 0.000 description 19
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 14
- 108010093096 Immobilized Enzymes Proteins 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 7
- 238000010647 peptide synthesis reaction Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 102100026066 Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 Human genes 0.000 description 6
- 238000000354 decomposition reaction Methods 0.000 description 6
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- 238000000926 separation method Methods 0.000 description 6
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 5
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- 102000004169 proteins and genes Human genes 0.000 description 5
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- 239000002994 raw material Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- -1 Alkylene glycol Chemical compound 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000003431 cross linking reagent Substances 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 3
- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 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
- 238000000502 dialysis Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 2
- 241000590035 Achromobacter lyticus Species 0.000 description 2
- 239000006171 Britton–Robinson buffer Substances 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- 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 2
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- VPUXQKZOFJQNLL-KRWDZBQOSA-N n-[(2s)-6-amino-1-(4-nitroanilino)-1-oxohexan-2-yl]benzamide Chemical compound N([C@@H](CCCCN)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)C(=O)C1=CC=CC=C1 VPUXQKZOFJQNLL-KRWDZBQOSA-N 0.000 description 2
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- BZCVUUOCIRSZBE-WGTBXUJISA-N (2S)-6-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[2-[[(1R,6R,12S,15S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,50S,53S,56S,59S,62S,65S,68S,71S,74R,77S,80S,83S,88R)-88-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[(2-aminoacetyl)amino]-3-methylpentanoyl]amino]-3-methylbutanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]-6-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-amino-3-phenylpropanoyl]amino]-3-methylbutanoyl]amino]-4-oxobutanoyl]amino]-5-oxopentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-47-[[(1S)-3-amino-1-carboxy-3-oxopropyl]carbamoyl]-53-(2-amino-2-oxoethyl)-62-(3-amino-3-oxopropyl)-24,56-bis(2-carboxyethyl)-12,71,80-tris(hydroxymethyl)-33,50,65-tris[(4-hydroxyphenyl)methyl]-15-(1H-imidazol-5-ylmethyl)-27,83-dimethyl-18,30,36,59,68-pentakis(2-methylpropyl)-7,10,13,16,19,22,25,28,31,34,37,40,49,52,55,58,61,64,67,70,73,76,79,82,85,87-hexacosaoxo-21,39,77-tri(propan-2-yl)-3,4,44,45,90,91-hexathia-8,11,14,17,20,23,26,29,32,35,38,41,48,51,54,57,60,63,66,69,72,75,78,81,84,86-hexacosazabicyclo[72.11.7]dononacontane-42-carbonyl]amino]acetyl]amino]-4-carboxybutanoyl]amino]-5-carbamimidamidopentanoyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]hexanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)CN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H]1CSSC[C@@H]2NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CSSC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc3cnc[nH]3)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)Cc3ccccc3)C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](Cc3cnc[nH]3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc3ccc(O)cc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](Cc3ccc(O)cc3)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc3ccc(O)cc3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC2=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](Cc2ccccc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H]([C@@H](C)O)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCCCN)C(O)=O)NC1=O)C(C)C BZCVUUOCIRSZBE-WGTBXUJISA-N 0.000 description 1
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- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N o-dimethylbenzene Natural products CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229920001281 polyalkylene Polymers 0.000 description 1
- 229920001748 polybutylene Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
本発明は新規な修飾酵素に関する。
更に詳しくは、アクロモバクター・リテイカス
から産生されるリシルエンドペプチダーゼ(アク
ロモバクター・プロテアーゼ)(以下、PLPを
略称する。)をポリアルキレングリコールを架橋
剤として用いて、分子間で架橋重合させて得られ
る新規な修飾酵素に関する。
酸素反応は一般に広く利用されているが、通
常、酵素は水性溶液に溶解した状態で使用される
為、反応終了後の酵素回収は至難であると共に、
反応生成物の分離精製も容易ではない。更に元
来、酵素は決して安定ではなく、通常中性域で常
温付近、常圧下に使用されるが、回収によつて著
しく損耗する。従つて、広い温度域及び広いPH域
で安定、各種の溶剤中に於いても使用出来、簡単
に回収できるという要件を備えることは、全ての
酵素に共通した志向であり、PLPに於いても当
然の要求であつた。
PLPは土壌から分離されたアクロモバクタ
ー・リテイカスM497−1が産生する菌外酵素で、
副島、正木らによつて発見され、アグリカルチユ
ラル・アンド・バイオロジカル・ケミストリー、
第42巻1443頁(1978年)、及び特開昭54−119085
号に記載されているアクロモバクター・プロテア
ーゼと同一の酵素である。至適温度は45℃、カ
ゼインを基質とした場合、プロテアーゼ活性の至
適PHは8.5〜10.5、ゲル過法による分子量は
27000、等電点PH6.9、ジイソプロピルフオスフオ
クロリド及びトシル−L−リシンクロロメチルケ
トンによつて強く阻害を受け、トシル−L−フエ
ニルアラニンクロロメチルケトン、エチレンジア
ミンテトラアセテート、オルソフエナンスロリン
及びp−クロロマーキユリーベンゾエートで阻害
されないセリン酵素であり、L−リジンのカルボ
キシル基に於けるエステル結合及びアミド結合を
特異的に水解し、それ故にアミノ酸配列決定の際
のペプチドの酵素分解並びにリシル−ペプチドの
分解及び合成に於いて有用な酵素であることは周
知の如くである。近年、この酵素を利用して豚イ
ンシユリンからヒトインシユリンの合成が行なわ
れているが(副島・正木らの特開昭54−135789号
及び森原・岡らのバイオケミカル・アンド・バイ
オフイジカル・リサーチ・コミユニケイシヨン、
92巻、362頁(1980年))、これらの方法はいずれ
も水溶性酵素単体を使用する液相反応であるた
め、得られたヒトインシユリンと異質の蛋白であ
るPLPを分離精製する必要があり、もし精製が
不十分である場合は、医薬品として人体に投与し
た場合に異種蛋白による抗体の生成という免疫学
的な副作用を判ない、殊に長期間に連続的に投与
する場合は極めて重大な結果を惹起する恐れがあ
る。更に当然の如く分離精製には多大の労力と時
間を要し、又分離後の酵素は失活の為、再使用に
耐えないので、高価な酵素を製造バツチ毎に使い
捨てることになり、その経済的損失は大なるもの
がある。それ故本酵素を何らかの誘導性に導き、
安定性を増し、且つ反応液からの分離を容易に行
なえる形態にして使用すれば、目的物の品質向上
並びに経済面からも優位性を保ち得ることは明ら
かである。
一般にこのような目的を達成する為、酵素を誘
導体に導く方法としては数多くの方法が知られて
いるが、それらの方法の内、代表的な方法を列挙
すると、(1)酵素を水不溶性の担体に共有結合させ
る方法、(2)酵素を水不溶性の担体にイオン結合さ
せる方法、(3)酵素を水不溶性の担体に吸着させる
方法、(4)酵素を架橋剤で架橋させ、水に不溶性な
ものにする方法、(5)担体を重合させ、酵素を包括
させ水に不溶なものにする方法、などがある。
しかしながら、上記の方法で得られた所謂、固
体化酵素は、水に不溶性であるために、バツチ法
で種々の反応に使用するに際して、反応液が不均
一な懸濁状態になつたり、あるいは比重が高い固
定化酵素はすぐに沈澱してしまうために、反応を
速やかに進行させる場合には、可能な限り反応液
を撹拌させたり、あるいは反応容器を回転させた
りする事により出来るだけ均一な懸濁状態を作
り、固定化酵素と反応物との接触時間を長くした
り、あるいは接触頻度を多くする必要がある。従
つて、反応させる物質が非常に不安定であり、空
気中の酸素により酸化され易く、激しい機械的な
撹拌又は回転は避けなければならないような反応
には固定化酵素は使用出来ない場合もある。
また、水に不溶性の固定化酵素を用いた反応に
於ては、当然のことながら、固相と液相との不均
一な反応となる為、液相中での反応に比較すると
必然的に反応速度が遅くなることは否めない事実
である。
更に、カラム法及びバツチ法に於いて、水不溶
性固定化酵素を反応に用いる場合、基質は溶媒に
溶解させているが、反応中に生じてくる反応生成
物あるいは副生成物のいずれか又は両者ともが、
用いる溶媒に難溶性もしくは不溶性の時には、固
定化酵素との分離が非常に困難になつてくる。沈
澱として生じてくる反応生成物と固定化酵素とを
分離するために、数多くの方法が試みられている
が、例えば、沈澱を生じせしめないような有機溶
媒などを使用する方法に於ては、有機溶剤の種類
によつては、時には著しく固定化酵素の失活を招
き、その結果、繰り返し使用回数も減少し、経済
性も悪くなる。また、反応生成物が反応溶媒に溶
解する場合であつても、繰り返し使用している間
に、反応生成物が水に不溶性な固定化酵素に非特
異的な吸着を惹起し、目的物の収率が悪くなる欠
点もある。
また、繰り返し固定化酵素を使用するに従い、
その担体の比活性が徐々に低下するため、反応率
を上昇させるには、当然新しい固定化酵素を補充
せねばならないが、バツチ法に於いて反応を行な
う場合、基質濃度との関係から溶媒の量を増加で
きない事もあり、このような時には実際には反応
が不可能になる。
本発明者らは、固定化酵素のかかる問題点を解
決すずべく、水に可溶で、尚且つ原料及び得られ
た生成物との分離が容易な修飾酵素について鋭意
研究を重ねた結果、ポリアルキレングリコール
(以下、PAGと略称する。)架橋剤として用いて、
PLPを分子間で架橋重合させることにより、酵
素活性を減ずることなく、不溶性を保つたまま
で、安定で、且つ原料及び反応生成物との分離が
容易な、新規な修飾酵素が得られることを見出
し、本発明を完成するに到つた。
これまで酵素療法の分野で、その免疫原性を低
下させることを目的として、分子表面に存在する
アミノ酸残基に非免疫原性高分子を化学結合さ
せ、これにより酵素分子表面に存在する抗原決定
部位を覆いかくし、抗体との結合能を減少あるい
は消失させる為に、酵素タンパクを合成高分子、
例えば、モノメトキシポリエチレングリコールの
ような一価性のポリエチレングリコールで化学修
飾する試みがなされている例があるが、この場合
はあくまでも酵素タンパクのもつ免疫原性を低下
させることが目的であつて、その為に一価性のポ
リエチレングリコール即ち、モノメトキシポリエ
チレングリコールでその周囲を覆うわけである
が、本発明の如く、不溶性を保つたままで酵素を
更に高分子化し、酵素反応に用いられる基質や反
応生成物との分離を容易ならしめる目的で、架橋
剤として二価性のポリアルキレングリコールを用
いて酵素を架橋重合させている例はこれまでに無
く、本発明者らが独自の知見に基いて完成した新
規な発明である。
本発明の修飾酵素即ち、ポリアルキレングリコ
ール−リシルエンドペプチダーゼ重合体(以下、
PAG−PLP重合体と略称する。)は、PAG分子
中の水酸基を活性化剤で活性化後、次いでこれに
PLPを作用させることにより製造される。
本発明に使用されるポリアルキレングリコール
としては、ポリエチレングリコール(以下、
PEGと略称する。)、ポリプロピレングリコール、
ポリブチレングリコール等が挙げられ、いずれも
両端の水酸基がメトキシ基に置換されていない二
価性のものが用いられる。また、PEGの場合に
は通常、分子量200〜20000のものが用いられる
が、好ましくは分子量6000〜20000のものがよい。
本発明の修飾酵素を得る方法は、好ましくは2
工程で行なわれる。先ず、PAGに活性化剤を反
応させて活性化させる。活性化剤としては、例え
ば1,1′−カルボニルジイミダゾール、塩化シア
ヌル等が使用され、これらはPAG中の水酸基1
個に対し1〜10倍量を用いるのが好ましい。反応
は一般にジオキサンやベンゼンなどの有機溶剤中
で行なわれ、温度は0〜50℃で行なうのが好まし
い。
このようにして活性化されたPAGは、一旦反
応液から単離・精製したのち、第2の反応を行な
うのが好ましい。活性化PAGの単離は、反応後、
透析により未反応の低分子量のものを除去する
か、ゲル過するか、限外過するか、もしくは
有機溶媒による沈澱法を用いるのが良い結果を与
える。第2工程は、活性化されたPAGとPLPと
を水溶液中、好ましくは弱アルカリ性の条件下で
反応させる。活性化PAGの量は、PLP分子中の
アミノ基の数に対し1〜1000倍量になるようにす
る。反応は0〜40℃で行なわれ、塩化シアヌルで
活性化されたPAGを用いた場合は1〜5時間で、
1,1′−カルボニルジイミダゾールで活性化され
たPAGを用いた場合は1〜5日間で完結する。
反応後の分離・精製は、水溶性高分子化合物、特
に蛋白質の精製法として汎用されている方法、例
えば透析法・ゲル過・限外過法・有機溶剤に
よる沈澱法などを適宜選択して利用する事ができ
る。
PLPの反応液中での最終濃度は、通常0.02〜20
重量%、より好ましくは0.1〜15重量%に調製さ
れる。酵素濃度が低すぎると、目的物である水溶
性PAG−PLP重合体の収率が低く、実用性に乏
しくなる。また、酵素濃度が高すぎると、得られ
てくる重合体が水不溶性となる恐れがある。
本発明に使用される活性化PAGは、二官能基
もしくはそれ以上の官能基を有するものであれば
いずれでもよいが、通常は先に挙げた1,1′−カ
ルボニルジイミダゾール又は塩化シアヌルで活性
化された二官能基、若しくは三官能基を有する活
性化PAGが用いられる。
本発明のPAG−PLPは、重合前のPLPと基質
特異性に於て変らず、また、至適PH、至適温度、
ミカエリス定数(Km値)及び最大速度Vmaxに関
して、重合前のPLPの値と変化がなかつた。
例えば、PEG−PLPの基質特異性を第1表に
示す。
The present invention relates to novel modified enzymes. More specifically, lysyl endopeptidase (Achromobacter protease) (hereinafter abbreviated as PLP) produced from Achromobacter lyticus is intermolecularly crosslinked and polymerized using polyalkylene glycol as a crosslinking agent. The present invention relates to the novel modified enzyme obtained. Oxygen reactions are widely used in general, but enzymes are usually used dissolved in an aqueous solution, so it is extremely difficult to recover the enzymes after the reaction is completed.
Separation and purification of reaction products is also not easy. Furthermore, enzymes are by no means stable, and are normally used in the neutral range at room temperature and under normal pressure, but they are subject to significant loss during recovery. Therefore, the requirements common to all enzymes are to be stable over a wide temperature range and pH range, to be usable in various solvents, and to be easily recovered. It was a natural request. PLP is an extrabacterial enzyme produced by Achromobacter lyticus M497-1 isolated from soil.
Discovered by Soejima, Masaki et al., agricultural and biological chemistry,
Volume 42, page 1443 (1978), and Japanese Patent Publication No. 119085 (1978)
It is the same enzyme as Achromobacter protease described in No. The optimum temperature is 45℃, the optimum pH for protease activity is 8.5 to 10.5 when casein is used as a substrate, and the molecular weight by gel filtration method is
27000, isoelectric point PH6.9, strongly inhibited by diisopropylphosphochloride and tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, ethylenediaminetetraacetate, orthophenanthroline and It is a serine enzyme that is not inhibited by p-chloromercury benzoate, and specifically hydrolyzes the ester and amide bonds in the carboxyl group of L-lysine, thus preventing enzymatic degradation of peptides and lysyl-lysine during amino acid sequencing. It is well known that enzymes are useful in the decomposition and synthesis of peptides. In recent years, human insulin has been synthesized from porcine insulin using this enzyme (see JP-A-54-135789 by Soejima and Masaki et al. and Biochemical and Biophysical Research by Morihara and Oka et al. Comic Union,
(Vol. 92, p. 362 (1980)). Since all of these methods are liquid-phase reactions that use a water-soluble enzyme alone, it is necessary to separate and purify the obtained human insulin and PLP, which is a foreign protein. If the purification is insufficient, the immunological side effect of producing antibodies due to foreign proteins cannot be detected when administered to the human body as a drug, and especially when administered continuously over a long period of time, this can lead to extremely serious consequences. There is a risk of causing Furthermore, it goes without saying that separation and purification requires a great deal of effort and time, and since the enzyme after separation is inactivated, it cannot be reused. The economic loss will be huge. Therefore, it leads to some kind of inducibility of this enzyme,
It is clear that if it is used in a form that increases stability and can be easily separated from the reaction solution, it will be advantageous in terms of improving the quality of the target product and economically. In general, many methods are known for deriving enzymes into derivatives in order to achieve this purpose. Among these methods, the most representative methods are: (1) converting enzymes into water-insoluble derivatives; (2) method of ionically bonding the enzyme to a water-insoluble carrier; (3) method of adsorbing the enzyme to a water-insoluble carrier; (4) method of cross-linking the enzyme with a cross-linking agent to make it insoluble in water. (5) polymerizing a carrier to entrap the enzyme and making it insoluble in water. However, since the so-called solidified enzyme obtained by the above method is insoluble in water, when it is used in various reactions in the batch method, the reaction solution may become a non-uniform suspension state or the specific gravity may vary. Immobilized enzymes with a high concentration precipitate quickly, so if you want the reaction to proceed quickly, it is best to stir the reaction solution as much as possible or rotate the reaction vessel to make the suspension as uniform as possible. It is necessary to create a turbid state, prolong the contact time between the immobilized enzyme and the reactant, or increase the frequency of contact. Therefore, immobilized enzymes may not be used in reactions where the substances to be reacted are extremely unstable and easily oxidized by oxygen in the air, and where vigorous mechanical stirring or rotation must be avoided. . In addition, in reactions using immobilized enzymes that are insoluble in water, naturally the reaction is heterogeneous between the solid phase and the liquid phase, so compared to reactions in the liquid phase, it is inevitable that It is an undeniable fact that the reaction rate is slow. Furthermore, in the column method and batch method, when a water-insoluble immobilized enzyme is used in the reaction, the substrate is dissolved in the solvent, but the reaction products and/or by-products generated during the reaction are Tomoga,
When the enzyme is poorly soluble or insoluble in the solvent used, it becomes extremely difficult to separate it from the immobilized enzyme. Many methods have been tried to separate the reaction product produced as a precipitate from the immobilized enzyme, but for example, methods using organic solvents that do not cause precipitates, Depending on the type of organic solvent, the immobilized enzyme may sometimes be significantly deactivated, resulting in a decrease in the number of times of repeated use and poor economic efficiency. Furthermore, even if the reaction product is dissolved in the reaction solvent, during repeated use, the reaction product may cause nonspecific adsorption to the water-insoluble immobilized enzyme, resulting in the recovery of the target product. There is also the drawback that the rate is poor. Additionally, as the immobilized enzyme is repeatedly used,
Since the specific activity of the carrier gradually decreases, it is naturally necessary to replenish new immobilized enzyme to increase the reaction rate. However, when carrying out the reaction in a batch method, the solvent In some cases, the amount cannot be increased, and in such cases, the reaction is actually impossible. In order to solve these problems with immobilized enzymes, the present inventors have conducted intensive research on modified enzymes that are soluble in water and can be easily separated from raw materials and obtained products. Alkylene glycol (hereinafter abbreviated as PAG) is used as a crosslinking agent,
We have discovered that by intermolecular cross-linking polymerization of PLP, a novel modified enzyme can be obtained that is stable, maintains insolubility, and is easy to separate from raw materials and reaction products without reducing enzyme activity. , we have completed the present invention. Until now, in the field of enzyme therapy, the aim of reducing the immunogenicity of enzyme therapy is to chemically bond non-immunogenic polymers to amino acid residues present on the surface of the enzyme molecule, thereby determining the antigen present on the surface of the enzyme molecule. In order to cover the site and reduce or eliminate its ability to bind to antibodies, the enzyme protein is coated with synthetic polymers,
For example, there have been attempts to chemically modify the enzyme protein with monovalent polyethylene glycol such as monomethoxypolyethylene glycol, but in this case, the purpose was to reduce the immunogenicity of the enzyme protein. For this purpose, the surrounding area is covered with monovalent polyethylene glycol, that is, monomethoxypolyethylene glycol. However, as in the present invention, the enzyme is further polymerized while remaining insoluble, and the substrate used in the enzyme reaction and the reaction To date, there has been no example of cross-linking and polymerizing enzymes using divalent polyalkylene glycol as a cross-linking agent for the purpose of facilitating separation from the product, and the present inventors have developed this method based on their own knowledge. This is a completed new invention. The modified enzyme of the present invention, namely polyalkylene glycol-lysyl endopeptidase polymer (hereinafter referred to as
It is abbreviated as PAG-PLP polymer. ), after activating the hydroxyl group in the PAG molecule with an activating agent,
Manufactured by reacting with PLP. The polyalkylene glycol used in the present invention includes polyethylene glycol (hereinafter referred to as
It is abbreviated as PEG. ), polypropylene glycol,
Examples include polybutylene glycol, and divalent ones in which the hydroxyl groups at both ends are not substituted with methoxy groups are used. Further, in the case of PEG, those with a molecular weight of 200 to 20,000 are usually used, but those with a molecular weight of 6,000 to 20,000 are preferable. The method for obtaining the modified enzyme of the present invention preferably includes two
It is done in the process. First, PAG is activated by reacting with an activator. As the activator, for example, 1,1'-carbonyldiimidazole, cyanuric chloride, etc. are used, and these
It is preferable to use 1 to 10 times the amount. The reaction is generally carried out in an organic solvent such as dioxane or benzene, preferably at a temperature of 0 to 50°C. It is preferable to perform the second reaction after the PAG activated in this manner is once isolated and purified from the reaction solution. Isolation of activated PAG is carried out after the reaction.
Good results can be obtained by removing unreacted low molecular weight substances by dialysis, gel filtration, ultrafiltration, or precipitation using an organic solvent. In the second step, activated PAG and PLP are reacted in an aqueous solution, preferably under slightly alkaline conditions. The amount of activated PAG is 1 to 1000 times the number of amino groups in the PLP molecule. The reaction was carried out at 0-40°C and for 1-5 hours using cyanuric chloride activated PAG.
When PAG activated with 1,1'-carbonyldiimidazole is used, the process is completed in 1 to 5 days.
For separation and purification after the reaction, use methods commonly used to purify water-soluble polymer compounds, especially proteins, such as dialysis, gel filtration, ultrafiltration, precipitation with organic solvents, etc., as appropriate. I can do that. The final concentration of PLP in the reaction solution is usually 0.02-20
It is adjusted to % by weight, more preferably 0.1 to 15% by weight. If the enzyme concentration is too low, the yield of the target water-soluble PAG-PLP polymer will be low, making it impractical. Furthermore, if the enzyme concentration is too high, the resulting polymer may become water-insoluble. The activated PAG used in the present invention may be any one having a difunctional group or more, but it is usually activated with the above-mentioned 1,1'-carbonyldiimidazole or cyanuric chloride. An activated PAG having a difunctional group or a trifunctional group is used. The PAG-PLP of the present invention has the same substrate specificity as PLP before polymerization, and also has an optimum pH, an optimum temperature,
Regarding Michaelis constant (Km value) and maximum velocity Vmax, there was no change from the value of PLP before polymerization. For example, the substrate specificity of PEG-PLP is shown in Table 1.
【表】
尚、上記酵素類の活性測定法は次のように行な
つた。即ち、0.2M2−アミノ−2−メチル−1,
3−プロパンジオール緩衝液(PH9.5)2.6mlに、
2.5mM各基質溶液0.3mlを加え、30℃にて予備加
温後、酵素溶液0.1mlを添加し、各基質に応じて
10分間から20時間、30℃にて反応させた。反応
後、45%酢酸水溶液10mlを加え反応を停止させた
のち、405nmにて比色測定し、その吸光度を求め
た。酵素単位としては、30℃に於いて1分間当り
1μmoleのp−ニトロアニリンを生成する酵素量
を1単位とした。酵素力価の算出方法は、次式に
従つた。(p−ニトロアニリンの分子吸光係数は、
9.62×103l・mole-1・cm-1である。)
活性(u/ml)=ΔO.D./min×1/9.62×4.0/0.1・
希釈倍数
第1表から明らかなように、本発明のPEG−
PLP重合体はリジンのカルボキシ基のアミド結
合に強く作用し、アルギニンに対しては殆んど作
用せず、またその他のアミノ酸残基のアミド結合
を加水分解することはなく、重合前のPLPと同
じ基質特異性を示すものである。
本発明の酵素誘導体の大きな特徴の1つは、分
子量が大きいために、ゲル過法によつて目的の
反応生成物と酵素とを非常に容易に分離でき、ま
た回収できた本発明PAG−PLP重合体は、その
酵素活性に全く損耗をきたしていないので、繰り
返し使用する事ができる点である。即ち、PLP
は分子量が27000〜30000であるため、分子量10万
以下のペプチドあるいは蛋白とのゲル過法によ
る完全な分離は容易ではなく、他の、より複雑な
方法を用いる必要があるが、本発明の酵素誘導体
は、分子量40万以上と極めて高分子であるので容
易に分離ができ、蛋白やペプチドの分解、合成の
生成物と酵素との分離・精製に極めて利する所大
なるものがある。
以上の如く、本発明の修飾酵素は、修飾前の酵
素と基質特異性、至適PH、至適温度、ミカエリス
定数(Km値)、最大速度Vmax共に変らず、酵素
活性を減ずることなく、水溶性を保つたままで高
分子化されており、安定で、反応液からの分離が
容易な為、反応生成物中に混入して問題を生じる
恐れもなく、又、酵素自体のくり返し再使用も容
易にできる点等に大きな特徴を有するものである
が、更に、本発明の修飾酵素の意外な効果として
は、修飾した結果、有機溶剤に対する溶解性が著
しく増大した点が挙げられる。本効果を示すもの
として、次に、本発明PEG−PLP重合体の各種
有機溶剤に対する溶解性データを第2表に示す。[Table] The activity of the enzymes mentioned above was measured as follows. That is, 0.2M2-amino-2-methyl-1,
Add 2.6 ml of 3-propanediol buffer (PH9.5),
Add 0.3ml of 2.5mM each substrate solution, prewarm at 30℃, add 0.1ml of enzyme solution, and adjust according to each substrate.
The reaction was carried out at 30°C for 10 minutes to 20 hours. After the reaction, 10 ml of 45% acetic acid aqueous solution was added to stop the reaction, and the absorbance was determined by colorimetric measurement at 405 nm. The enzyme unit is per minute at 30°C.
The amount of enzyme that produces 1 μmole of p-nitroaniline was defined as 1 unit. The enzyme titer was calculated according to the following formula. (The molecular extinction coefficient of p-nitroaniline is
It is 9.62×10 3 l・mole -1・cm -1 . ) Activity (u/ml) = ΔO.D./min×1/9.62×4.0/0.1・
Dilution factor As is clear from Table 1, the PEG-
PLP polymer strongly acts on the amide bond of the carboxyl group of lysine, has almost no effect on arginine, and does not hydrolyze the amide bond of other amino acid residues, and is similar to PLP before polymerization. They exhibit the same substrate specificity. One of the major features of the enzyme derivative of the present invention is that, due to its large molecular weight, the desired reaction product and enzyme can be separated and recovered very easily by gel filtration. The advantage of the polymer is that it can be used repeatedly, as there is no loss in its enzymatic activity. That is, PLP
has a molecular weight of 27,000 to 30,000, so it is not easy to completely separate it from peptides or proteins with a molecular weight of 100,000 or less by gel filtration, and other, more complicated methods must be used. Derivatives are extremely high molecules with a molecular weight of 400,000 or more, so they can be easily separated, and are extremely useful in the decomposition of proteins and peptides, and in the separation and purification of synthetic products and enzymes. As described above, the modified enzyme of the present invention has the same substrate specificity, optimum pH, optimum temperature, Michaelis constant (Km value), and maximum velocity Vmax as the enzyme before modification, does not reduce enzyme activity, and is water soluble. Since it is polymerized while maintaining its properties, it is stable and easy to separate from the reaction solution, so there is no risk of it getting mixed into the reaction product and causing problems, and the enzyme itself can be easily reused repeatedly. In addition, an unexpected effect of the modified enzyme of the present invention is that the solubility in organic solvents is significantly increased as a result of modification. As an indication of this effect, Table 2 shows the solubility data of the PEG-PLP polymer of the present invention in various organic solvents.
【表】【table】
【表】
また第2表に於ける有機溶剤に溶解された本品
の溶液を、30℃にて200分間加温した後、アミダ
ーゼ活性の測定を行ない、水に溶解させたものと
の活性の比較を行なつた結果を第3表に示す。[Table] In addition, the amidase activity of the solution of this product dissolved in the organic solvent shown in Table 2 was heated at 30°C for 200 minutes, and the activity was compared with that dissolved in water. Table 3 shows the results of the comparison.
【表】【table】
【表】
第3表から明らかなように、重合前のPLPは
ジメチルスルホキシドとベンゼンにしか溶解せ
ず、また溶解しても殆んど活性が残つていない
が、一方、PEG−PLP重合体は、アセトン中で
は失活はなく、PEG6−PLPに於てはむしろ活性
が増大しており、またジオキサン、ベンゼン、ク
ロロホルム及びテトラヒドロフラン中ではかなり
の活性を保持している。
このようにPAG−PLPの重合体は、水素では
勿論の事、有機溶剤系に於いても反応に使用する
ことが可能である。従来、酵素反応は酵素の溶解
性の故に水の存在下で行なうのが通常の方法であ
り、有機溶済下では実施できないというのが常識
であつたが、本発明の酵素誘導体をもつてすれ
ば、酵素反応を利用した各種技術分野での新しい
展開が期待できる。
例えば化学法によるペプチド合成は、一般に熱
や圧力をかけるためラセミ化が起こり易く、光学
純度が下がる欠点があり、また、圧力をかける場
合には特別の装置を必要とする。
一方、酵素を用いるペプチド合成は、一般には
常温・常圧での反応のため、ラセミ化が起こりに
くく、且つ酵素の基質特異性が高いために、ラセ
ミ体の原料を用いてもいずれか一方の原料としか
反応せず光学分割が容易であるので、光学純度の
高いものが得られる。
一般に、ペプチド合成は、反応機構及び使用す
る原料や反応生成物の溶解性の面からすれば、有
機溶剤中で反応を行なうのが好ましいが、酵素法
の場合、反応に用いる酵素の溶解性及び活性の発
現の面から、反応は水溶液中で行なわざるを得
ず、水と混和する有機溶剤を用いる場合でも、有
機溶剤の濃度は可能な限り低い方がよいとされて
おり、一般的には65%以下の濃度で使用されてい
る。
従つて、酵素によるペプチド合成は、ペプチド
合成の反応機構からは有機溶剤を高濃度に使用す
るのがよいとされているが、他方、酵素を触媒に
用いるためには有機溶剤を出来るだけ低濃度に抑
える必要があるという二律背反の問題から生じる
種々の制約を受けることになる。
しかしながら、本発明者らは、本発明の酵素誘
導体、即ちPAG−PLP重合体の有機溶剤中での
溶解性に着目し、95容量%という高濃度の有機溶
剤中、PEG−PLP重合体を用いてペプチド合成
を試みた結果、ペプチド合成が高収率で進行する
という驚くべき事実をも見い出している。即ち、
ジメチルホルムアミド85容量%、エタノール10容
量%及び0.5Mトリス−塩酸緩衝液(PH6.5)5容
量%中、PEG6−PLP重合体0.95単位存在下、デ
スアラニンインスリン20mg(3.5mM)とスレオ
ニン第3級ブチルエステル90mg(340mM)とを
37℃で15時間反応させ、反応液を高速液体クロマ
トグラフイーにより分析したところ、B30位スレ
オニン第3級ブチルエステルインスリンが81%の
変換率で得られていることが確認された。
この場合、有機溶剤としてジメチルホルムアミ
ド及びエタノールを用いているが、これらの溶剤
に溶かしたPEG−PLP重合体は、第3表に示さ
れているようにペプチドの加水分解に於いては、
あまり活性が残つていなかつたものであるが、ペ
プチド合成に於いては、意外にも酵素活性を示し
ており、また、回収したPEG6−PLP重合体は、
その酵素活性に全く損耗をきたしていなかつた。
このように溶解能の高い有機溶剤系でのPAG
−PLP重合体の利用は、リジンを含むペプチド
の合成及び水解に於いて有効な手段となりうるこ
と自明である。
本発明の修飾酵素、即ちPAG−PLP重合体は
水溶性であるため、均一系で反応できること及び
極めて安定性が高いため、酵素活性を損うことな
く、回収及び繰り返し使用が可能である事、水単
独でも、水と混和できる有機溶剤を広範囲に併用
する系でも、高収率に酵素反応が行なわれる事、
更に本発明PAG−PLP重合体は、高分子量であ
るため、反応系から例えば分子篩ゲル過法等に
より簡単に取り出すことができるので、目的物の
分離・精製も、また本発明PAG−PLP重合体の
回収も極めて有利である事から、本発明の利用価
値は非常に高く、具体的には、アミノ酸配列決定
の際のペプチドの酵素分解、並びにリシル−ペプ
チドの分解及び合成など幅広い目的に利用でき、
斯業に貢献するところ甚だ大なるものがある。
以下に参考例及び実施例を挙げて本発明を更に
詳しく説明するが、本発明はこれらによつて何ら
限定されるものでないことはいうまでもない。
参考例 1
200ml容コルベン中、PEG6000(平均分子量
7500、ライオン油脂製)15gをジオキサン50mlに
溶解し、1,1′−カルボニルジイミダゾール4.05
gを添加溶解後、37℃にて2時間反応させた。反
応後、反応液を透析チユーブ(スペクトラポア
6、分画分子量3500、スペクトラム・メデイカ
ル・インダストリー社製)に入れ、水に対して一
夜透析後、透析内液を凍結乾燥させ、活性化
PEG6000(以下、活性化PEG6と略称する。)を
14.5g得た。
参考例 2
塩化シアヌル5.5g(0.03mole)を、無水炭酸
ナトリウム10gを含有する無水ベンゼン400mlに
溶解し、PEG20000(平均分子量20000±5000、日
本油脂製)100g(0.005mole)を加えた後、室
温で一夜反応後、反応混合物を過し、液に石
油エーテル600mlをゆつくり撹拌しながら加えた。
析出する沈澱を取後、沈澱をベンゼン400mlに
再溶解し、石油エーテル600mlを撹拌下ゆつくり
加え、析出する沈澱を取する。同様に沈澱析出
工程及び過工程を数回繰り返し、活性化
PEG20000(以下、活性化PEG20と略称する。)が
80g得られた。
実施例 1
PLP3.6単位を10mMホウ酸塩緩衝液(PH8.5)
0.5mlに溶解後、参考例1で得られた活性化PEG6
を338mg添加溶解し、4℃にて72時間撹拌反応さ
せた。反応後、反応液をセフアデツクスG−100
(フアルマシア社製)のカラム(φ1.5×85cm)に
よるゲル過に付し、カラム容積比0.36の位置に
PEG6−PLP重合体が2.92単位(収率81.1%)得
られた。ここに得られた重合体の諸性質は以下の
通りである。
(1) 分子量
40万以上(セフアデツクスG−200を用いるゲ
ル過法による)
(2) PH安定性
緩衝液としてブリトン・ロビンソン緩衝液(PH
2〜12)を用い、30℃にて1時間保持した後、本
重合体の活性を測定した結果、本重合体はPH5〜
11で安定である事が認められた。(第1図参照)
(3) 熱安定性
0.2M2−アミノ−2−メチル−1,3−プロパ
ンジオール緩衝液(PH9.5)を用いて、各温度で
1時間加温後の結果、本重合体は40℃まで安定で
ある事が認められた。(第2図参照)
(4) ミカエリス定数
0.2M2−アミノ−2−メチル−1,3−プロパ
ンジオール緩衝液(PH9.5)中、30℃にてベンゾ
イル・リジン−p−ニトロアニリドを基質とした
場合、本重合体のKm値は0.17mMであつた。また
最大速度Vmaxは0.19μmole/min/mlであつた。
(5) 至適PH
ブリトン・ロビンソン緩衝液(PH3〜12)を用
いて、30℃にて各PHに於ける本重合体の活性測定
を行なつた結果、本重合体の至適PHは9.5であつ
た。
(6) 至適温度
0.2M2−アミノ−2−メチル−1,3−プロパ
ンジオール緩衝液(PH9.5)中、ベンゾイル・リ
ジン−p−ニトロアニリドを基質とし、各温度に
於ける活性を測定した結果、本重合体の至適温度
は50℃であつた。
(7) 保存安定性
10mMホウ酸塩緩衝液(PH8.5)中、4℃にて
40日間保存後の活性をチエツクした結果、活性の
低下はなく、またセフアデツクスG−100のカラ
ムによるゲル過では、本重合体の分解も認めら
れなかつた。
実施例 2
PLP2.8単位と、参考例2で得られた活性化
PEG20300mgとを100mMホウ酸塩緩衝液(PH9.2)
0.5mlに溶解し、4℃にて1時間撹拌反応させた
後、実施例1と同様に処理を行ない、PEG20−
PLP重合体が1.58単位(収率56.4%)得られた。
ここに得られた本重合体の諸性質を以下に示す。
(測定法は実施例1の場合と同様に行なつた。)
(1) 分子量:40万以上。
(2) PH安定性:PH5〜11に於いて安定(第1図参
照)。
(3) 熱安定性:40℃迄安定(第2図参照)
(4) ミカエリス定数(Km値):0.18mM
最大速度Vmax:0.18μmole/min/ml
(5) 至適PH:PH9.5〜10.0
(6) 至適温度:50℃
(7) 保存安定性:失活及び分解なし[Table] As is clear from Table 3, PLP before polymerization dissolves only in dimethyl sulfoxide and benzene, and even after dissolution, almost no activity remains; on the other hand, PEG-PLP polymer is not inactivated in acetone, but rather has increased activity in PEG 6 -PLP, and retains considerable activity in dioxane, benzene, chloroform and tetrahydrofuran. In this way, the PAG-PLP polymer can be used for reactions not only in hydrogen but also in organic solvents. Conventionally, it was common knowledge that enzymatic reactions were carried out in the presence of water due to the solubility of enzymes, and that they could not be carried out in organic solutions. For example, we can expect new developments in various technological fields that utilize enzyme reactions. For example, peptide synthesis by chemical methods generally has the disadvantage that racemization easily occurs due to the application of heat and pressure, resulting in a decrease in optical purity.Additionally, when pressure is applied, special equipment is required. On the other hand, in peptide synthesis using enzymes, racemization is difficult to occur because the reaction is generally carried out at room temperature and pressure, and the substrate specificity of the enzyme is high, so even if racemic raw materials are used, either one of them will not occur. Since it reacts only with raw materials and is easy to optically resolve, products with high optical purity can be obtained. In general, peptide synthesis is preferably carried out in an organic solvent from the viewpoint of the reaction mechanism and the solubility of the raw materials and reaction products used, but in the case of enzymatic methods, the solubility of the enzyme used in the reaction In order to express activity, the reaction must be carried out in an aqueous solution, and even when using an organic solvent that is miscible with water, it is said that the concentration of the organic solvent should be as low as possible; Used at concentrations below 65%. Therefore, in peptide synthesis using enzymes, it is recommended to use organic solvents at high concentrations from the reaction mechanism of peptide synthesis, but on the other hand, in order to use enzymes as catalysts, organic solvents should be used at as low concentrations as possible. They are subject to various constraints arising from the trade-off between the two. However, the present inventors focused on the solubility of the enzyme derivative of the present invention, that is, the PAG-PLP polymer, in organic solvents, and used the PEG-PLP polymer in an organic solvent at a high concentration of 95% by volume. As a result of trying to synthesize peptides, they discovered the surprising fact that peptide synthesis proceeds with high yield. That is,
20 mg (3.5 mM) of desalanine insulin and 20 mg of threonine insulin in the presence of 0.95 units of PEG 6 -PLP polymer in 85 vol. % dimethylformamide, 10 vol. % ethanol, and 5 vol. % 0.5 M Tris-HCl buffer (PH 6.5). 90mg (340mM) of tertiary butyl ester
The reaction was carried out at 37° C. for 15 hours, and the reaction solution was analyzed by high performance liquid chromatography, and it was confirmed that B 30 -threonine tertiary butyl ester insulin was obtained at a conversion rate of 81%. In this case, dimethylformamide and ethanol are used as organic solvents, and as shown in Table 3, the PEG-PLP polymer dissolved in these solvents has the following effects in the hydrolysis of peptides:
Although it did not have much activity remaining, it unexpectedly showed enzymatic activity in peptide synthesis, and the recovered PEG 6 -PLP polymer
There was no loss in the enzyme activity. PAG in organic solvent systems with high solubility
- It is obvious that the use of PLP polymers can be an effective means for the synthesis and hydrolysis of lysine-containing peptides. The modified enzyme of the present invention, that is, the PAG-PLP polymer, is water-soluble, so it can be reacted in a homogeneous system, and it is extremely stable, so it can be recovered and used repeatedly without loss of enzyme activity. Enzyme reactions can be carried out in high yields with water alone or with a wide range of water-miscible organic solvents.
Furthermore, since the PAG-PLP polymer of the present invention has a high molecular weight, it can be easily removed from the reaction system by, for example, molecular sieve gel filtration. Since the recovery of lysyl peptides is also extremely advantageous, the utility value of the present invention is extremely high. Specifically, it can be used for a wide range of purposes such as enzymatic decomposition of peptides during amino acid sequencing, and decomposition and synthesis of lysyl peptides. ,
There is a tremendous amount to contribute to this industry. The present invention will be explained in more detail below with reference to Reference Examples and Examples, but it goes without saying that the present invention is not limited thereto. Reference example 1 PEG6000 (average molecular weight
7500, manufactured by Lion Oil) in 50 ml of dioxane, 1,1'-carbonyldiimidazole 4.05
After adding and dissolving g, the mixture was reacted at 37°C for 2 hours. After the reaction, the reaction solution was placed in a dialysis tube (Spectrapore 6, molecular weight cutoff 3500, manufactured by Spectrum Medical Industries), dialyzed against water overnight, and the dialyzed solution was freeze-dried and activated.
PEG6000 (hereinafter abbreviated as activated PEG 6 )
I got 14.5g. Reference Example 2 5.5 g (0.03 mole) of cyanuric chloride was dissolved in 400 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate, and 100 g (0.005 mole) of PEG20000 (average molecular weight 20000±5000, manufactured by NOF) was added, and the mixture was heated to room temperature. After reacting overnight, the reaction mixture was filtered, and 600 ml of petroleum ether was slowly added to the solution with stirring.
After removing the precipitate, redissolve the precipitate in 400 ml of benzene, slowly add 600 ml of petroleum ether with stirring, and remove the precipitate. Similarly, repeat the precipitation step and overstep several times to activate
PEG20000 (hereinafter abbreviated as activated PEG 20 )
80g was obtained. Example 1 3.6 units of PLP in 10mM borate buffer (PH8.5)
After dissolving in 0.5ml, activated PEG 6 obtained in Reference Example 1
338 mg was added and dissolved, and the mixture was stirred and reacted at 4°C for 72 hours. After the reaction, transfer the reaction solution to Sephadex G-100.
(manufactured by Pharmacia Co., Ltd.) column (φ1.5 x 85 cm), and the column volume ratio was 0.36.
2.92 units (81.1% yield) of PEG 6 -PLP polymer were obtained. The properties of the polymer obtained here are as follows. (1) Molecular weight 400,000 or more (by gel filtration method using Sephadex G-200) (2) PH stability Britton-Robinson buffer (PH
As a result of measuring the activity of this polymer after holding it at 30℃ for 1 hour using
11 and was found to be stable. (See Figure 1) (3) Thermal stability After heating at each temperature for 1 hour using 0.2M 2-amino-2-methyl-1,3-propanediol buffer (PH9.5), this The polymer was found to be stable up to 40°C. (See Figure 2) (4) Michaelis constant Benzoyl lysine-p-nitroanilide was used as a substrate in 0.2M 2-amino-2-methyl-1,3-propanediol buffer (PH9.5) at 30°C. In this case, the Km value of this polymer was 0.17mM. Moreover, the maximum speed Vmax was 0.19 μmole/min/ml. (5) Optimal PH As a result of measuring the activity of this polymer at each PH at 30°C using Britton-Robinson buffer (PH 3 to 12), the optimal PH of this polymer was 9.5. It was hot. (6) Optimum temperature Measure the activity at each temperature using benzoyl lysine-p-nitroanilide as a substrate in 0.2M 2-amino-2-methyl-1,3-propanediol buffer (PH9.5). As a result, the optimum temperature for this polymer was 50°C. (7) Storage stability In 10mM borate buffer (PH8.5) at 4℃
As a result of checking the activity after storage for 40 days, there was no decrease in activity, and no decomposition of the polymer was observed by gel filtration using a Sephadex G-100 column. Example 2 PLP2.8 units and activation obtained in Reference Example 2
300mg of PEG 20 and 100mM borate buffer (PH9.2)
After dissolving in 0.5 ml and reacting with stirring at 4°C for 1 hour, the same treatment as in Example 1 was carried out to obtain PEG 20 -
1.58 units of PLP polymer (yield 56.4%) were obtained.
The properties of the obtained polymer are shown below.
(The measurement method was the same as in Example 1.) (1) Molecular weight: 400,000 or more. (2) PH stability: Stable at PH5-11 (see Figure 1). (3) Thermal stability: Stable up to 40℃ (see Figure 2) (4) Michaelis constant (Km value): 0.18mM Maximum speed Vmax: 0.18μmole/min/ml (5) Optimal PH: PH9.5~ 10.0 (6) Optimum temperature: 50℃ (7) Storage stability: No deactivation or decomposition
第1図は、実施例1及び実施例2に於ける本発
明のPEG6−PLP重合体及びPEG20−PLP重合体
のPH安定性を示す曲線を、また第2図は、同じく
熱安定性を示す曲線を表わす。
FIG. 1 shows the curves showing the PH stability of the PEG 6 -PLP polymer and PEG 20 -PLP polymer of the present invention in Example 1 and Example 2, and FIG. represents a curve showing
Claims (1)
ンドペプチダーゼ(アクロモバクター・プロテア
ーゼ)をポリアルキレングリコールで架橋重合
させて得られる修飾酵素。 2 ポリアルキレングリコールが、1,1′−カル
ボニルジイミダゾールで活性化されたポリアルキ
レングリコールである、特許請求の範囲第1項記
載の修飾酵素。 3 ポリアルキレングリコールが、塩化シアヌル
で活性化されたポリアルキレングリコールであ
る、特許請求の範囲第1項記載の修飾酵素。 4 ポリアルキレングリコールがポリエチレング
リコールである、特許請求の範囲第1項、第2項
又は第3項記載の修飾酵素。 5 ポリエチレングリコールの分子量が200から
20000である特許請求の範囲第4項記載の修飾酵
素。[Scope of Claims] 1. A modified enzyme obtained by cross-linking and polymerizing lysyl endopeptidase produced by Achromobacter reticus (Achromobacter protease) with polyalkylene glycol. 2. The modified enzyme according to claim 1, wherein the polyalkylene glycol is a polyalkylene glycol activated with 1,1'-carbonyldiimidazole. 3. The modified enzyme according to claim 1, wherein the polyalkylene glycol is a polyalkylene glycol activated with cyanuric chloride. 4. The modified enzyme according to claim 1, 2 or 3, wherein the polyalkylene glycol is polyethylene glycol. 5 The molecular weight of polyethylene glycol is from 200
20,000.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22822083A JPS60120985A (en) | 1983-12-02 | 1983-12-02 | Novel modification enzyme |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22822083A JPS60120985A (en) | 1983-12-02 | 1983-12-02 | Novel modification enzyme |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60120985A JPS60120985A (en) | 1985-06-28 |
JPH0476673B2 true JPH0476673B2 (en) | 1992-12-04 |
Family
ID=16873053
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP22822083A Granted JPS60120985A (en) | 1983-12-02 | 1983-12-02 | Novel modification enzyme |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60120985A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014061344A1 (en) | 2012-10-15 | 2014-04-24 | 信越化学工業株式会社 | Waterproof sheet for outdoor tank and method for applying same |
-
1983
- 1983-12-02 JP JP22822083A patent/JPS60120985A/en active Granted
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014061344A1 (en) | 2012-10-15 | 2014-04-24 | 信越化学工業株式会社 | Waterproof sheet for outdoor tank and method for applying same |
Also Published As
Publication number | Publication date |
---|---|
JPS60120985A (en) | 1985-06-28 |
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