JPH11290078A - Yeast having lipase on cell surface and its utilization - Google Patents
Yeast having lipase on cell surface and its utilizationInfo
- Publication number
- JPH11290078A JPH11290078A JP10097937A JP9793798A JPH11290078A JP H11290078 A JPH11290078 A JP H11290078A JP 10097937 A JP10097937 A JP 10097937A JP 9793798 A JP9793798 A JP 9793798A JP H11290078 A JPH11290078 A JP H11290078A
- Authority
- JP
- Japan
- Prior art keywords
- yeast
- sequence
- lipase
- cell surface
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 97
- 108090001060 Lipase Proteins 0.000 title claims abstract description 70
- 239000004367 Lipase Substances 0.000 title claims abstract description 69
- 102000004882 Lipase Human genes 0.000 title claims abstract description 68
- 235000019421 lipase Nutrition 0.000 title claims abstract description 68
- 210000004027 cell Anatomy 0.000 title claims abstract description 62
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 51
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 27
- 101710204899 Alpha-agglutinin Proteins 0.000 claims abstract description 13
- 241000146406 Fusarium heterosporum Species 0.000 claims abstract description 5
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 29
- 239000000194 fatty acid Substances 0.000 claims description 29
- 229930195729 fatty acid Natural products 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 25
- 150000004665 fatty acids Chemical class 0.000 claims description 19
- 239000013612 plasmid Substances 0.000 claims description 13
- 230000003248 secreting effect Effects 0.000 claims description 13
- 150000001413 amino acids Chemical class 0.000 claims description 12
- -1 fatty acid ester Chemical class 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 210000004899 c-terminal region Anatomy 0.000 claims description 11
- 230000004807 localization Effects 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000000910 agglutinin Substances 0.000 claims description 6
- 230000001070 adhesive effect Effects 0.000 claims description 5
- 239000000853 adhesive Substances 0.000 claims description 4
- 230000028327 secretion Effects 0.000 abstract description 10
- 239000002699 waste material Substances 0.000 abstract description 7
- 239000003225 biodiesel Substances 0.000 abstract description 4
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 150000002632 lipids Chemical class 0.000 abstract 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 80
- 239000003921 oil Substances 0.000 description 33
- 235000019198 oils Nutrition 0.000 description 33
- 239000003925 fat Substances 0.000 description 27
- 235000019197 fats Nutrition 0.000 description 26
- 108020004414 DNA Proteins 0.000 description 24
- 238000006243 chemical reaction Methods 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- 239000012634 fragment Substances 0.000 description 12
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 235000019626 lipase activity Nutrition 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000004220 aggregation Methods 0.000 description 6
- 230000002776 aggregation Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000004382 Amylase Substances 0.000 description 5
- 102000013142 Amylases Human genes 0.000 description 5
- 108010065511 Amylases Proteins 0.000 description 5
- 229920002472 Starch Polymers 0.000 description 5
- 235000019418 amylase Nutrition 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 239000008107 starch Substances 0.000 description 5
- 235000019698 starch Nutrition 0.000 description 5
- 238000005809 transesterification reaction Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108091092724 Noncoding DNA Proteins 0.000 description 4
- 229920005830 Polyurethane Foam Polymers 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000446 fuel Substances 0.000 description 4
- 230000003100 immobilizing effect Effects 0.000 description 4
- 239000011496 polyurethane foam Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 102220201851 rs143406017 Human genes 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 3
- 241000235070 Saccharomyces Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000014593 oils and fats Nutrition 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 239000002344 surface layer Substances 0.000 description 3
- BITHHVVYSMSWAG-KTKRTIGZSA-N (11Z)-icos-11-enoic acid Chemical compound CCCCCCCC\C=C/CCCCCCCCCC(O)=O BITHHVVYSMSWAG-KTKRTIGZSA-N 0.000 description 2
- YWWVWXASSLXJHU-AATRIKPKSA-N (9E)-tetradecenoic acid Chemical compound CCCC\C=C\CCCCCCCC(O)=O YWWVWXASSLXJHU-AATRIKPKSA-N 0.000 description 2
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 101710139410 1-phosphatidylinositol phosphodiesterase Proteins 0.000 description 2
- XDOFQFKRPWOURC-UHFFFAOYSA-N 16-methylheptadecanoic acid Chemical compound CC(C)CCCCCCCCCCCCCCC(O)=O XDOFQFKRPWOURC-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- 101710186708 Agglutinin Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 2
- 102100022624 Glucoamylase Human genes 0.000 description 2
- 101710146024 Horcolin Proteins 0.000 description 2
- RQJUKVXWAKJDBW-SVSWQMSJSA-N Ile-Ser-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N RQJUKVXWAKJDBW-SVSWQMSJSA-N 0.000 description 2
- 101710189395 Lectin Proteins 0.000 description 2
- 101710179758 Mannose-specific lectin Proteins 0.000 description 2
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 2
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- MKGIILKDUGDRRO-FXQIFTODSA-N Pro-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 MKGIILKDUGDRRO-FXQIFTODSA-N 0.000 description 2
- 102100037097 Protein disulfide-isomerase A3 Human genes 0.000 description 2
- 235000021355 Stearic acid Nutrition 0.000 description 2
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 2
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 2
- RPECVQBNONKZAT-WZLNRYEVSA-N Thr-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H]([C@@H](C)O)N RPECVQBNONKZAT-WZLNRYEVSA-N 0.000 description 2
- 230000004523 agglutinating effect Effects 0.000 description 2
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000002551 biofuel Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 210000001822 immobilized cell Anatomy 0.000 description 2
- 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 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- 229960004488 linolenic acid Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 2
- FBUKVWPVBMHYJY-UHFFFAOYSA-N nonanoic acid Chemical compound CCCCCCCCC(O)=O FBUKVWPVBMHYJY-UHFFFAOYSA-N 0.000 description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- 235000021313 oleic acid Nutrition 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- SECPZKHBENQXJG-FPLPWBNLSA-N palmitoleic acid Chemical compound CCCCCC\C=C/CCCCCCCC(O)=O SECPZKHBENQXJG-FPLPWBNLSA-N 0.000 description 2
- CNVZJPUDSLNTQU-SEYXRHQNSA-N petroselinic acid Chemical compound CCCCCCCCCCC\C=C/CCCCC(O)=O CNVZJPUDSLNTQU-SEYXRHQNSA-N 0.000 description 2
- 108010031719 prolyl-serine Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000008117 stearic acid Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- OGILYBDMVOATLU-CQJMVLFOSA-N (2s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-n-[(2s)-1-[[(2s)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]-4-methylpentanamide Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)C1=CC=C(O)C=C1 OGILYBDMVOATLU-CQJMVLFOSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- OXEDXHIBHVMDST-UHFFFAOYSA-N 12Z-octadecenoic acid Natural products CCCCCC=CCCCCCCCCCCC(O)=O OXEDXHIBHVMDST-UHFFFAOYSA-N 0.000 description 1
- ZONJATNKKGGVSU-UHFFFAOYSA-N 14-methylpentadecanoic acid Chemical compound CC(C)CCCCCCCCCCCCC(O)=O ZONJATNKKGGVSU-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- YWWVWXASSLXJHU-UHFFFAOYSA-N 9E-tetradecenoic acid Natural products CCCCC=CCCCCCCCC(O)=O YWWVWXASSLXJHU-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 1
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 1
- RAAWHFXHAACDFT-FXQIFTODSA-N Ala-Met-Asn Chemical compound CSCC[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CC(N)=O)C(O)=O RAAWHFXHAACDFT-FXQIFTODSA-N 0.000 description 1
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 1
- WQKAQKZRDIZYNV-VZFHVOOUSA-N Ala-Ser-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WQKAQKZRDIZYNV-VZFHVOOUSA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 1
- NLYYHIKRBRMAJV-AEJSXWLSSA-N Ala-Val-Pro Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N NLYYHIKRBRMAJV-AEJSXWLSSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- NXDXECQFKHXHAM-HJGDQZAQSA-N Arg-Glu-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NXDXECQFKHXHAM-HJGDQZAQSA-N 0.000 description 1
- VLDRQOHCMKCXLY-SRVKXCTJSA-N Asn-Ser-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VLDRQOHCMKCXLY-SRVKXCTJSA-N 0.000 description 1
- NJIKKGUVGUBICV-ZLUOBGJFSA-N Asp-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O NJIKKGUVGUBICV-ZLUOBGJFSA-N 0.000 description 1
- ZRUBWRCKIVDCFS-XPCJQDJLSA-N Asp-Leu-Thr-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZRUBWRCKIVDCFS-XPCJQDJLSA-N 0.000 description 1
- XMKXONRMGJXCJV-LAEOZQHASA-N Asp-Val-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XMKXONRMGJXCJV-LAEOZQHASA-N 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 235000021357 Behenic acid Nutrition 0.000 description 1
- DPUOLQHDNGRHBS-UHFFFAOYSA-N Brassidinsaeure Natural products CCCCCCCCC=CCCCCCCCCCCCC(O)=O DPUOLQHDNGRHBS-UHFFFAOYSA-N 0.000 description 1
- 101100083069 Candida albicans (strain SC5314 / ATCC MYA-2876) PGA62 gene Proteins 0.000 description 1
- 101100106993 Candida albicans (strain SC5314 / ATCC MYA-2876) YWP1 gene Proteins 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- 235000002568 Capsicum frutescens Nutrition 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 108090000746 Chymosin Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- URXZXNYJPAJJOQ-UHFFFAOYSA-N Erucic acid Natural products CCCCCCC=CCCCCCCCCCCCC(O)=O URXZXNYJPAJJOQ-UHFFFAOYSA-N 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150054379 FLO1 gene Proteins 0.000 description 1
- 101150006710 FLO8 gene Proteins 0.000 description 1
- CITDWMLWXNUQKD-FXQIFTODSA-N Gln-Gln-Asn Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CITDWMLWXNUQKD-FXQIFTODSA-N 0.000 description 1
- AFODTOLGSZQDSL-PEFMBERDSA-N Glu-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N AFODTOLGSZQDSL-PEFMBERDSA-N 0.000 description 1
- FGGKGJHCVMYGCD-UKJIMTQDSA-N Glu-Val-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGGKGJHCVMYGCD-UKJIMTQDSA-N 0.000 description 1
- 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 1
- DWUKOTKSTDWGAE-BQBZGAKWSA-N Gly-Asn-Arg Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DWUKOTKSTDWGAE-BQBZGAKWSA-N 0.000 description 1
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- YKRYHWJRQUSTKG-KBIXCLLPSA-N Ile-Ala-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKRYHWJRQUSTKG-KBIXCLLPSA-N 0.000 description 1
- HDODQNPMSHDXJT-GHCJXIJMSA-N Ile-Asn-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O HDODQNPMSHDXJT-GHCJXIJMSA-N 0.000 description 1
- NCSIQAFSIPHVAN-IUKAMOBKSA-N Ile-Asn-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N NCSIQAFSIPHVAN-IUKAMOBKSA-N 0.000 description 1
- XLXPYSDGMXTTNQ-DKIMLUQUSA-N Ile-Phe-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(O)=O XLXPYSDGMXTTNQ-DKIMLUQUSA-N 0.000 description 1
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 1
- CIDLJWVDMNDKPT-FIRPJDEBSA-N Ile-Phe-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N CIDLJWVDMNDKPT-FIRPJDEBSA-N 0.000 description 1
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 1
- ZYVTXBXHIKGZMD-QSFUFRPTSA-N Ile-Val-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ZYVTXBXHIKGZMD-QSFUFRPTSA-N 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- OGCQGUIWMSBHRZ-CIUDSAMLSA-N Leu-Asn-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OGCQGUIWMSBHRZ-CIUDSAMLSA-N 0.000 description 1
- DBSLVQBXKVKDKJ-BJDJZHNGSA-N Leu-Ile-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DBSLVQBXKVKDKJ-BJDJZHNGSA-N 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- IUWMQCZOTYRXPL-ZPFDUUQYSA-N Lys-Ile-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O IUWMQCZOTYRXPL-ZPFDUUQYSA-N 0.000 description 1
- RPWTZTBIFGENIA-VOAKCMCISA-N Lys-Thr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O RPWTZTBIFGENIA-VOAKCMCISA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- 101100043636 Oryza sativa subsp. japonica SSIIIA gene Proteins 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000021319 Palmitoleic acid Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 239000005643 Pelargonic acid Substances 0.000 description 1
- CNVZJPUDSLNTQU-UHFFFAOYSA-N Petroselaidic acid Natural products CCCCCCCCCCCC=CCCCCC(O)=O CNVZJPUDSLNTQU-UHFFFAOYSA-N 0.000 description 1
- WFDAEEUZPZSMOG-SRVKXCTJSA-N Phe-Cys-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O WFDAEEUZPZSMOG-SRVKXCTJSA-N 0.000 description 1
- AKJAKCBHLJGRBU-JYJNAYRXSA-N Phe-Glu-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N AKJAKCBHLJGRBU-JYJNAYRXSA-N 0.000 description 1
- BFYHIHGIHGROAT-HTUGSXCWSA-N Phe-Glu-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BFYHIHGIHGROAT-HTUGSXCWSA-N 0.000 description 1
- SHUFSZDAIPLZLF-BEAPCOKYSA-N Phe-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)O SHUFSZDAIPLZLF-BEAPCOKYSA-N 0.000 description 1
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- SUENWIFTSTWUKD-AVGNSLFASA-N Pro-Leu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SUENWIFTSTWUKD-AVGNSLFASA-N 0.000 description 1
- JDJMFMVVJHLWDP-UNQGMJICSA-N Pro-Thr-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JDJMFMVVJHLWDP-UNQGMJICSA-N 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- 101100066910 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FLO1 gene Proteins 0.000 description 1
- 101100066911 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FLO5 gene Proteins 0.000 description 1
- 241001125046 Sardina pilchardus Species 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- WTUJZHKANPDPIN-CIUDSAMLSA-N Ser-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N WTUJZHKANPDPIN-CIUDSAMLSA-N 0.000 description 1
- PZZJMBYSYAKYPK-UWJYBYFXSA-N Ser-Ala-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O PZZJMBYSYAKYPK-UWJYBYFXSA-N 0.000 description 1
- HBZBPFLJNDXRAY-FXQIFTODSA-N Ser-Ala-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O HBZBPFLJNDXRAY-FXQIFTODSA-N 0.000 description 1
- YMEXHZTVKDAKIY-GHCJXIJMSA-N Ser-Asn-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO)C(O)=O YMEXHZTVKDAKIY-GHCJXIJMSA-N 0.000 description 1
- XWCYBVBLJRWOFR-WDSKDSINSA-N Ser-Gln-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O XWCYBVBLJRWOFR-WDSKDSINSA-N 0.000 description 1
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 1
- GWMXFEMMBHOKDX-AVGNSLFASA-N Ser-Gln-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 GWMXFEMMBHOKDX-AVGNSLFASA-N 0.000 description 1
- UOLGINIHBRIECN-FXQIFTODSA-N Ser-Glu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UOLGINIHBRIECN-FXQIFTODSA-N 0.000 description 1
- GZBKRJVCRMZAST-XKBZYTNZSA-N Ser-Glu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZBKRJVCRMZAST-XKBZYTNZSA-N 0.000 description 1
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 1
- ZUDXUJSYCCNZQJ-DCAQKATOSA-N Ser-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CO)N ZUDXUJSYCCNZQJ-DCAQKATOSA-N 0.000 description 1
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 0.000 description 1
- MQQBBLVOUUJKLH-HJPIBITLSA-N Ser-Ile-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MQQBBLVOUUJKLH-HJPIBITLSA-N 0.000 description 1
- XXNYYSXNXCJYKX-DCAQKATOSA-N Ser-Leu-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O XXNYYSXNXCJYKX-DCAQKATOSA-N 0.000 description 1
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 1
- BYCVMHKULKRVPV-GUBZILKMSA-N Ser-Lys-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O BYCVMHKULKRVPV-GUBZILKMSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- FLONGDPORFIVQW-XGEHTFHBSA-N Ser-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FLONGDPORFIVQW-XGEHTFHBSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- WUXCHQZLUHBSDJ-LKXGYXEUSA-N Ser-Thr-Asp Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC(O)=O)C(O)=O WUXCHQZLUHBSDJ-LKXGYXEUSA-N 0.000 description 1
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- ANOQEBQWIAYIMV-AEJSXWLSSA-N Ser-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N ANOQEBQWIAYIMV-AEJSXWLSSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- 101150094640 Siae gene Proteins 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- BSNZTJXVDOINSR-JXUBOQSCSA-N Thr-Ala-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O BSNZTJXVDOINSR-JXUBOQSCSA-N 0.000 description 1
- JMZKMSTYXHFYAK-VEVYYDQMSA-N Thr-Arg-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O JMZKMSTYXHFYAK-VEVYYDQMSA-N 0.000 description 1
- PQLXHSACXPGWPD-GSSVUCPTSA-N Thr-Asn-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PQLXHSACXPGWPD-GSSVUCPTSA-N 0.000 description 1
- GKMYGVQDGVYCPC-IUKAMOBKSA-N Thr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)N GKMYGVQDGVYCPC-IUKAMOBKSA-N 0.000 description 1
- YSXYEJWDHBCTDJ-DVJZZOLTSA-N Thr-Gly-Trp Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O YSXYEJWDHBCTDJ-DVJZZOLTSA-N 0.000 description 1
- JQAWYCUUFIMTHE-WLTAIBSBSA-N Thr-Gly-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JQAWYCUUFIMTHE-WLTAIBSBSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- BDGBHYCAZJPLHX-HJGDQZAQSA-N Thr-Lys-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BDGBHYCAZJPLHX-HJGDQZAQSA-N 0.000 description 1
- SPVHQURZJCUDQC-VOAKCMCISA-N Thr-Lys-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O SPVHQURZJCUDQC-VOAKCMCISA-N 0.000 description 1
- WNQJTLATMXYSEL-OEAJRASXSA-N Thr-Phe-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O WNQJTLATMXYSEL-OEAJRASXSA-N 0.000 description 1
- VTMGKRABARCZAX-OSUNSFLBSA-N Thr-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O VTMGKRABARCZAX-OSUNSFLBSA-N 0.000 description 1
- STUAPCLEDMKXKL-LKXGYXEUSA-N Thr-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O STUAPCLEDMKXKL-LKXGYXEUSA-N 0.000 description 1
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 1
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 1
- KPMIQCXJDVKWKO-IFFSRLJSSA-N Thr-Val-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KPMIQCXJDVKWKO-IFFSRLJSSA-N 0.000 description 1
- AKHDFZHUPGVFEJ-YEPSODPASA-N Thr-Val-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AKHDFZHUPGVFEJ-YEPSODPASA-N 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- HVHJYXDXRIWELT-RYUDHWBXSA-N Tyr-Glu-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O HVHJYXDXRIWELT-RYUDHWBXSA-N 0.000 description 1
- YYLHVUCSTXXKBS-IHRRRGAJSA-N Tyr-Pro-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YYLHVUCSTXXKBS-IHRRRGAJSA-N 0.000 description 1
- QPZMOUMNTGTEFR-ZKWXMUAHSA-N Val-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N QPZMOUMNTGTEFR-ZKWXMUAHSA-N 0.000 description 1
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 1
- ZIGZPYJXIWLQFC-QTKMDUPCSA-N Val-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N)O ZIGZPYJXIWLQFC-QTKMDUPCSA-N 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- JAIZPWVHPQRYOU-ZJDVBMNYSA-N Val-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O JAIZPWVHPQRYOU-ZJDVBMNYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000010775 animal oil Substances 0.000 description 1
- 229940116226 behenic acid Drugs 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 108010066270 beta-lactorphin Proteins 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 229940080701 chymosin Drugs 0.000 description 1
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940108623 eicosenoic acid Drugs 0.000 description 1
- BITHHVVYSMSWAG-UHFFFAOYSA-N eicosenoic acid Natural products CCCCCCCCC=CCCCCCCCCCC(O)=O BITHHVVYSMSWAG-UHFFFAOYSA-N 0.000 description 1
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- DPUOLQHDNGRHBS-KTKRTIGZSA-N erucic acid Chemical compound CCCCCCCC\C=C/CCCCCCCCCCCC(O)=O DPUOLQHDNGRHBS-KTKRTIGZSA-N 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 1
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
- 229960002733 gamolenic acid Drugs 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 235000021299 gondoic acid Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 description 1
- 229940124736 hepatitis-B vaccine Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 description 1
- 229940073769 methyl oleate Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 229960002969 oleic acid Drugs 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 239000010773 plant oil Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920006327 polystyrene foam Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229940100486 rice starch Drugs 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 235000012045 salad Nutrition 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000003784 tall oil Substances 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 108010072641 thermostable lipase Proteins 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- AQWHMKSIVLSRNY-UHFFFAOYSA-N trans-Octadec-5-ensaeure Natural products CCCCCCCCCCCCC=CCCCC(O)=O AQWHMKSIVLSRNY-UHFFFAOYSA-N 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 235000019871 vegetable fat Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000010698 whale oil Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、細胞表面にリパー
ゼを有する酵母、その利用に関する。さらに詳しくは、
細胞表面にリパーゼを有する酵母を用いて、油脂の分解
あるいは生成を行う方法に関する。[0001] The present invention relates to a yeast having a lipase on a cell surface, and its use. For more information,
The present invention relates to a method for decomposing or producing fats and oils using yeast having a lipase on a cell surface.
【０００２】[0002]
【従来の技術】酵母はバイオテクノロジー産業にとって
重要な真核生物であり、アルコール発酵に代表される醸
造工業、あるいは、遺伝子組換え技術を用いて有用物質
（例えば、Ｂ型肝炎ワクチン、インスリン、あるいは酵
素キモシンなど）を生産、分泌するための宿主として用
いられている。2. Description of the Related Art Yeast is an important eukaryote for the biotechnology industry, and is a useful substance (for example, hepatitis B vaccine, insulin, Enzyme, chymosin, etc.).
【０００３】ところで酵母は、アミラーゼ、リパーゼな
どの分泌型酵素を有していない。そのため、酵母は澱粉
を分解して糖化することができず、澱粉を炭素源として
利用できない。そこで、酒作りにおいては、アミラーゼ
を分泌する麹菌等を用いて米の澱粉を糖化させ、つい
で、これに酵母を作用させて、アルコール発酵を行い、
酒を製造している。従って、酵母にアミラーゼをコード
する遺伝子を導入してアミラーゼを分泌させれば、酵母
は澱粉を唯一の炭素源として成育し、アルコール発酵が
可能となる。[0003] Incidentally, yeast does not have secretory enzymes such as amylase and lipase. Therefore, yeast cannot degrade and saccharify starch and cannot use starch as a carbon source. Therefore, in sake brewing, rice starch is saccharified using koji molds that secrete amylase, and then yeast is allowed to act on the starch to perform alcohol fermentation.
Manufactures sake. Therefore, if a gene encoding amylase is introduced into yeast to secrete amylase, the yeast grows using starch as a sole carbon source, and alcohol fermentation becomes possible.
【０００４】しかし、例えば、アミラーゼを分泌するよ
りも、酵母の細胞表層に固定化すれば、より効率的にア
ルコール発酵が可能となる。本発明者らは、グルコアミ
ラーゼを酵母表層に固定化して、澱粉を唯一の炭素源と
して生育し、アルコールを生産することに成功している
（植田、田中ら、Applied and Environmental Microbio
logy, 63: 1362-1366 (1997)）。However, for example, alcohol fermentation can be performed more efficiently if immobilized on the cell surface of yeast rather than secreting amylase. The present inventors have succeeded in immobilizing glucoamylase on a yeast surface, growing starch as a sole carbon source, and producing alcohol (Ueda, Tanaka et al., Applied and Environmental Microbio
logy, 63: 1362-1366 (1997)).
【０００５】一方、油脂の分野においては、環境問題な
どを背景に、石油燃料に代わって、油脂の脂肪酸を用い
る、いわゆるバイオディーゼル燃料の開発研究が行われ
ている。しかし、化学触媒を用いて油脂を分解し、脂肪
酸エステルを得るためには、比較的高温高圧で反応を行
わなければならず、エネルギー多消費型であるという欠
点がある。On the other hand, in the field of oils and fats, research and development of so-called biodiesel fuels using fatty acids of oils and fats in place of petroleum fuels are being conducted due to environmental problems and the like. However, in order to use a chemical catalyst to decompose fats and oils to obtain fatty acid esters, the reaction must be performed at a relatively high temperature and high pressure, which is disadvantageous in that it is energy-consuming.
【０００６】[0006]
【発明が解決しようとする課題】そこで、常温常圧で、
効率よくバイオ燃料を得る方法が求められていた。本発
明は、酵母の細胞表層にリパーゼを固定することによ
り、バイオ燃料を効率よく生産する方法を提供すること
を目的とする。本発明で得られる細胞表層にリパーゼを
有する酵母を、従来リパーゼが使用されていた種々の分
野、例えば、油脂のエステル交換、医薬品の中間体とし
て有用な光学異性体の分離、精製、あるいはエナンチオ
マー反転などに応用することを目的とする。Therefore, at normal temperature and normal pressure,
There has been a need for a method for efficiently obtaining biofuel. An object of the present invention is to provide a method for efficiently producing biofuel by immobilizing lipase on the cell surface of yeast. Yeast having lipase on the cell surface obtained by the present invention can be used in various fields in which lipase has been conventionally used, for example, transesterification of fats and oils, separation of optical isomers useful as intermediates of pharmaceuticals, purification, or inversion of enantiomer It is intended to be applied to such as.
【０００７】[0007]
【課題を解決するための手段】本発明は、分泌シグナル
配列、リパーゼの構造遺伝子配列、細胞表層局在タンパ
ク質の一部をコードする配列及びＧＰＩアンカー付着シ
グナル配列をこの順で有するＤＮＡであって、リパーゼ
を細胞表層に発現し得るＤＮＡに関する。The present invention provides a DNA comprising, in this order, a secretory signal sequence, a lipase structural gene sequence, a sequence encoding a part of a cell surface localization protein, and a GPI anchor attachment signal sequence. , Which can express lipase on the cell surface.
【０００８】好適な実施態様においては、前記細胞表層
局在タンパク質の一部をコードする配列及びアンカー付
着シグナル配列が酵母のα-アグルチニンの配列であ
る。In a preferred embodiment, the sequence encoding a part of the cell surface localization protein and the anchor attachment signal sequence are yeast α-agglutinin sequences.
【０００９】好適な実施態様においては、前記細胞表層
局在タンパク質の一部をコードする配列及びアンカー付
着シグナル配列が、α−アグルチニンのＣ末端から３２
０アミノ酸の配列である。[0009] In a preferred embodiment, the sequence encoding a part of the cell surface localization protein and the anchor attachment signal sequence are located at 32 from the C-terminal of α-agglutinin.
It is a sequence of 0 amino acids.
【００１０】より好適な実施態様においては、前記リパ
ーゼがFusarium heterosporum由来のリパーゼである。[0010] In a more preferred embodiment, the lipase is a lipase derived from Fusarium heterosporum.
【００１１】好適な実施態様においては、前記ＤＮＡが
プラスミドの形態である。[0011] In a preferred embodiment, the DNA is in the form of a plasmid.
【００１２】本発明は、また、リパーゼを細胞表層に有
する酵母であって、前記ＤＮＡを有する酵母に関する。[0012] The present invention also relates to a yeast having a lipase on a cell surface, the yeast having the DNA.
【００１３】好適な実施態様においては、前記酵母が、
担体に固定化されている。[0013] In a preferred embodiment, the yeast is
It is immobilized on a carrier.
【００１４】さらに、好適な実施態様においては、前記
酵母が凝集性または接着性の酵母である。Further, in a preferred embodiment, the yeast is a cohesive or adhesive yeast.
【００１５】本発明は、さらに、リパーゼを細胞表層に
有する酵母と油脂とを反応させる工程を含む、脂肪酸の
製造方法に関する。[0015] The present invention further relates to a method for producing a fatty acid, comprising a step of reacting a yeast having lipase on the cell surface with an oil or fat.
【００１６】本発明は、また、アルコールの存在下、リ
パーゼを細胞表層に有する酵母と油脂とを反応させる工
程を含む、脂肪酸エステルの製造方法に関する。The present invention also relates to a method for producing a fatty acid ester, comprising a step of reacting a yeast having a lipase on a cell surface with an oil or fat in the presence of an alcohol.
【００１７】本発明は、さらに、リパーゼを細胞表層に
有する酵母と油脂とを、必要に応じて脂肪酸の存在下、
反応させる工程を含む、油脂のエステル交換方法に関す
る。[0017] The present invention further comprises the step of combining a yeast having lipase on the cell surface with fats and oils, if necessary, in the presence of a fatty acid.
The present invention relates to a method for transesterifying fats and oils, comprising a step of reacting.
【００１８】[0018]
【発明の実施の形態】（本発明のＤＮＡ）本発明のＤＮ
Ａは、分泌シグナル配列、リパーゼの構造遺伝子配列、
細胞表層局在タンパク質の一部をコードする配列及びＧ
ＰＩアンカー付着シグナル配列をこの順で有するＤＮＡ
であって、リパーゼを細胞表層に発現し得るＤＮＡであ
る。その構造を図１に示す。BEST MODE FOR CARRYING OUT THE INVENTION (DNA of the present invention) DN of the present invention
A is a secretory signal sequence, a lipase structural gene sequence,
Sequence encoding a part of cell surface localized protein and G
DNA having a PI anchor attachment signal sequence in this order
And a DNA capable of expressing lipase on the cell surface. The structure is shown in FIG.
【００１９】分泌シグナルは、一般に細胞外（ペリプラ
ズムも含む）に分泌されるタンパク質（分泌性タンパク
質）のＮ-末端に結合している、疎水性に富んだアミノ
酸を多く含むアミノ酸配列であり、通常、分泌性タンパ
ク質が細胞内から細胞膜を通過して細胞外へ分泌される
際に除去される。The secretion signal is an amino acid sequence containing a large number of highly hydrophobic amino acids bound to the N-terminal of a protein (secretory protein) secreted extracellularly (including the periplasm). It is removed when secreted proteins are secreted from the inside of the cell, through the cell membrane and out of the cell.
【００２０】本発明においては、リパーゼを酵母の細胞
外に分泌（移動）させることができる分泌シグナルであ
れば、どのような分泌シグナルでも用いられ、起源は問
わない。例えば、分泌シグナル配列としては、リパーゼ
の分泌シグナル配列、酵母のα-またはａ-アグルチニン
のシグナル配列、グルコアミラーゼの分泌シグナル等が
好適に用いられる。リパーゼの活性に影響を与えないの
であれば、分泌シグナルの一部または全部がリパーゼの
Ｎ-末端に残ってもよい。In the present invention, any secretory signal can be used as long as it can secrete (migrate) the lipase out of the yeast cells, regardless of its origin. For example, as the secretion signal sequence, a lipase secretion signal sequence, a yeast α- or a-agglutinin signal sequence, a glucoamylase secretion signal, and the like are suitably used. Some or all of the secretion signal may remain at the N-terminus of the lipase as long as it does not affect the activity of the lipase.
【００２１】細胞表層局在タンパク質は、酵母の細胞表
層に固定され、細胞表層に存在するタンパク質をいう。
例えば、性凝集タンパク質であるα-またはａ-アグルチ
ニンが挙げられる。このようなタンパク質は、分泌シグ
ナルを有する点で分泌タンパク質と同様であるが、図２
に示すようにＧＰＩアンカーを介して細胞膜に固定され
て輸送される点で分泌タンパク質とは異なる。細胞表層
局在タンパク質はＣ末端にＧＰＩアンカー付着認識シグ
ナルを有しており、その認識配列を選択的に切断された
Ｃ末端部分でＧＰＩアンカーと結合して細胞膜に固定さ
れ、その後、PI-PLCにより、ＧＰＩアンカーの根元部が
切断され、細胞壁に組み込まれて細胞表層に固定され、
細胞表層に局在する。[0021] The cell surface localization protein refers to a protein that is immobilized on the yeast cell surface and is present on the cell surface.
For example, α- or a-agglutinin, which is a sex aggregation protein, can be mentioned. Such proteins are similar to secreted proteins in that they have a secretory signal.
As shown in Fig. 7, the protein is different from the secreted protein in that it is fixed to the cell membrane and transported via the GPI anchor. The cell surface localization protein has a GPI anchor attachment recognition signal at the C-terminus, and the recognition sequence is fixed to the cell membrane by binding to the GPI anchor at the selectively cleaved C-terminal portion. By this, the root of the GPI anchor is cut off, incorporated into the cell wall and fixed to the cell surface,
It is localized on the cell surface.
【００２２】ここで、ＧＰＩアンカーとは、glycosylph
osphatidylinositol(GPI)と呼ばれるエタノールアミン
リン酸-6マンノースα1-2マンノースα1-6マンノースα
1-4グルコサミンα1-6イノシトールリン脂質を基本構造
とする糖脂質をいい、PI-PLCとは、ホスファチジルイノ
シトール依存性ホスホリパーゼＣをいう。Here, the GPI anchor is glycosylph
Ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α called osphatidylinositol (GPI)
1-4 Glucosamine α1-6 refers to a glycolipid having a basic structure of inositol phospholipid, and PI-PLC refers to phosphatidylinositol-dependent phospholipase C.
【００２３】細胞表層局在タンパク質の一部をコードす
る配列とは、酵母の細胞表面に提示されるタンパク質
（例えばα-またはａ-アグルチニン）の一部をコードす
る配列をいい、主にＣ末端部分をコードする配列をいう
が、リパーゼの活性に悪影響を与えなければ、どのよう
な配列でもよい。好適には、α−アグルチニンのＣ末端
から３２０アミノ酸の配列をコードする配列が用いられ
る。このアミノ酸配列中には４個所の糖鎖結合部位があ
る。ＧＰＩアンカーがPI-PLCで切断された後に、この糖
鎖と細胞壁を構成する多糖類とが共有結合することによ
り、α-アグルチニンのＣ末端配列部分が細胞壁と結合
して保持されるので、特に有用である。The sequence encoding a part of the protein located on the cell surface layer refers to a sequence encoding a part of a protein (for example, α- or a-agglutinin) displayed on the cell surface of yeast, and mainly comprises a C-terminal. The term refers to a sequence encoding a portion, but may be any sequence as long as it does not adversely affect the activity of lipase. Preferably, a sequence encoding a sequence of 320 amino acids from the C-terminal of α-agglutinin is used. There are four sugar chain binding sites in this amino acid sequence. After the GPI anchor is cleaved by PI-PLC, the sugar chain and the polysaccharide constituting the cell wall are covalently bonded, whereby the C-terminal sequence portion of α-agglutinin is bound and retained in the cell wall. Useful.
【００２４】ＧＰＩアンカーシグナル配列とは、ＧＰＩ
アンカーが細胞表層局在タンパク質と結合する際に認識
される配列であり、通常、細胞表層局在タンパク質のＣ
末端あるいはその近傍に位置する配列である。酵母のα
-アグルチニン配列のＣ末端部分をコードする配列が好
適に用いられる。上記α−アグルチニンのＣ末端から３
２０アミノ酸の配列をコードする配列の３’末端側に
は、ＧＰＩアンカーシグナル配列をコードする配列が含
まれるので、このＣ末端から３２０アミノ酸の配列をコ
ードする配列が有用である。The GPI anchor signal sequence is a GPI anchor signal sequence.
The sequence recognized when the anchor binds to the cell surface-localized protein.
Sequence located at or near the end. Yeast α
-A sequence encoding the C-terminal part of the agglutinin sequence is preferably used. 3 from the C-terminal of the α-agglutinin
Since a sequence encoding a GPI anchor signal sequence is included at the 3 'end of a sequence encoding a sequence of 20 amino acids, a sequence encoding a sequence of 320 amino acids from the C-terminus is useful.
【００２５】油脂は、グリセロールに脂肪酸が結合した
ものをいい、本発明で「リパーゼ」とは、油脂から脂肪
酸を遊離し得る活性を有するタンパク質（酵素）をい
う。このような活性を有していれば、起源は問わない。
微生物、植物、および動物（例えば、豚膵臓）由来のリ
パーゼが用いられる。また、1,3-特異的であってもよい
し、非特異的であってもよい。用途により、適宜決定さ
れる。リパーゼとしては、例えば、J.Biochem.116巻、5
36-540(1994)に記載の、Fusarium heterosporum由来の
リパーゼが好適に用いられる。このリパーゼは、溶剤耐
性が高いので、油脂の分解、あるいはエステル交換反応
などの水分が少ない反応系で用いるのに特に有用であ
る。未知のリパーゼの遺伝子配列は、常法により、決定
すればよい。Fats and oils are those in which fatty acids are bound to glycerol. In the present invention, "lipase" refers to a protein (enzyme) having the activity of releasing fatty acids from fats and oils. The origin does not matter as long as it has such activity.
Lipases from microorganisms, plants, and animals (eg, pig pancreas) are used. In addition, it may be 1,3-specific or non-specific. It is appropriately determined according to the application. As lipase, for example, J. Biochem. 116, 5
The lipase derived from Fusarium heterosporum described in 36-540 (1994) is preferably used. Since this lipase has high solvent resistance, it is particularly useful for use in a reaction system with a small amount of water, such as decomposition of fats and oils or transesterification. The gene sequence of the unknown lipase may be determined by a conventional method.
【００２６】分泌シグナル配列、リパーゼの構造遺伝子
配列、細胞表層局在タンパク質の一部をコードする配列
及びＧＰＩアンカー付着シグナル配列をこの順で有する
ＤＮＡの合成は、当業者には周知の技術である。例え
ば、分泌シグナルとリパーゼの構造遺伝子との結合は、
部位特異的突然変異法を用いて行うことができ、正確な
分泌シグナルの切断と活性なリパーゼが発現できる。さ
らにこの配列と、細胞表層局在タンパク質の一部をコー
ドする配列及びＧＰＩアンカー付着シグナル配列とを結
合すればよい。結合は、適切な制限酵素、リンカー等を
用いて行うことができる。The synthesis of a DNA having a secretory signal sequence, a lipase structural gene sequence, a sequence encoding a part of a cell surface localization protein, and a GPI anchor attachment signal sequence in this order is a technique well known to those skilled in the art. . For example, the binding between the secretory signal and the lipase structural gene
This can be done using site-directed mutagenesis, which results in accurate cleavage of secretory signals and expression of active lipase. Furthermore, this sequence may be combined with a sequence encoding a part of a cell surface localization protein and a GPI anchor attachment signal sequence. The binding can be performed using an appropriate restriction enzyme, linker, or the like.
【００２７】本発明のＤＮＡはプラスミドの形態である
ことが望ましい。ＤＮＡの取得の簡易化の点からは、大
腸菌とのシャトルベクターであることが好ましい。本発
明のＤＮＡの出発材料としては、例えば、酵母の２μｍ
プラスミドの複製開始点(Ori）とColE1の複製開始点を
有しており、また、酵母選択マーカー（例えば、薬剤耐
性遺伝子、TRP、LEU2等）および大腸菌の選択マーカー
（薬剤耐性遺伝子等）を有することがさらに好ましい。
また、リパーゼ構造遺伝子を発現させるために、この遺
伝子の発現を調節するオペレーター、プロモーター、タ
ーミネーター、エンハンサー等のいわゆる調節配列をも
含んでいることが望ましい。例えば、GAPDH（グリセル
アルデヒド３’-リン酸デヒドロゲナーゼ）プロモータ
ーおよびGAPDHターミネーターが挙げられる。このよう
な出発材料のプラスミドとして、pYE22m、pYGA2270等が
挙げられる。The DNA of the present invention is preferably in the form of a plasmid. From the viewpoint of simplification of DNA acquisition, a shuttle vector with Escherichia coli is preferable. Starting materials for the DNA of the present invention include, for example, yeast 2 μm
It has a plasmid origin of replication (Ori) and a ColE1 origin of replication, and has a yeast selectable marker (eg, drug resistance gene, TRP, LEU2, etc.) and an E. coli selectable marker (eg, drug resistance gene). Is more preferable.
In addition, in order to express the lipase structural gene, it is desirable to include so-called regulatory sequences such as an operator, a promoter, a terminator, and an enhancer that regulate the expression of this gene. Examples include the GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) promoter and GAPDH terminator. Such starting material plasmids include pYE22m, pYGA2270, and the like.
【００２８】最も好適には、プラスミドpYGA2270又はpY
E2mのGAPDHプロモーターとGAPDHターミネーターの配列
の間に、分泌シグナル配列とリパーゼの構造遺伝子配列
とを有する配列とα-アグルチニンのＣ末端から３２０
アミノ酸をコードする配列とを結合した配列を挿入すれ
ば、本発明のＤＮＡが製造される。Most preferably, the plasmid pYGA2270 or pYGA2270
Between the sequence of the GAPDH promoter and the GAPDH terminator of E2m, a sequence having a secretory signal sequence and a structural gene sequence of lipase, and a sequence from the C-terminal of α-agglutinin of 320 ° C.
By inserting a sequence linked to a sequence encoding an amino acid, the DNA of the present invention is produced.
【００２９】宿主の酵母としては、凝集性の酵母が、固
定化して連続反応を行なう点から好ましい。As the host yeast, a cohesive yeast is preferable since it is immobilized and undergoes a continuous reaction.
【００３０】凝集性の酵母としては、Saccharomyces di
astaticus ATCC60715、同ATCC60712、Saccharomyces ce
revisiae IFO1953、同CG1945、同HF7Cが挙げられる。As cohesive yeast, Saccharomyces di
astaticus ATCC60715, ATCC60712, Saccharomyces ce
revisiae IFO1953, CG1945, and HF7C.
【００３１】（本発明の酵母の製造方法）本発明のリパ
ーゼを細胞表層に有する酵母は、本発明のＤＮＡを酵母
に導入することにより得られる。「ＤＮＡの導入」と
は、酵母の中にＤＮＡを導入し、発現させることを意味
する。ＤＮＡの導入は、形質転換、形質導入、トランス
フェクション、コトランスフェクション、エレクトロポ
レーション等の方法があり、具体的には、例えば、酢酸
リチウムを用いる方法、プロトプラスト法等がある。(Method for Producing Yeast of the Present Invention) The yeast having the lipase of the present invention on the cell surface can be obtained by introducing the DNA of the present invention into yeast. "Introduction of DNA" means that DNA is introduced into yeast and expressed. DNA can be introduced by methods such as transformation, transduction, transfection, co-transfection, and electroporation. Specific examples include a method using lithium acetate and a protoplast method.
【００３２】導入されるＤＮＡは、プラスミドの形態
で、あるいは宿主の遺伝子に挿入して、または宿主の遺
伝子と相同組換えを起こして染色体に取り込まれてもよ
い。The DNA to be introduced may be incorporated into a chromosome in the form of a plasmid, inserted into a host gene, or undergoing homologous recombination with the host gene.
【００３３】ＤＮＡが導入された酵母は、選択マーカー
（例えばTRP）で選択され、リパーゼ活性を測定するこ
とにより選択される。リパーゼが細胞表層に固定されて
いることは、抗リパーゼ抗体とFITC標識抗体を用いる免
疫抗体法で確認し得る。The yeast into which the DNA has been introduced is selected by using a selectable marker (eg, TRP), and is selected by measuring lipase activity. The immobilization of lipase on the cell surface can be confirmed by an immunoantibody method using an anti-lipase antibody and a FITC-labeled antibody.
【００３４】本発明の酵母は、担体に固定化されている
ことが、望ましい。連続反応あるいは回分式培養におい
て、繰り返し使用できるからである。The yeast of the present invention is preferably immobilized on a carrier. This is because it can be used repeatedly in a continuous reaction or batch culture.
【００３５】（細胞表層にリパーゼを有する固定化酵
母）「担体」とは、酵母を固定化することができる物質
を意味し、好ましくは、水またはある特定の溶媒に対し
て不溶性の物質である。本発明に用いる担体の材質とし
ては、例えば、ポリビニルアルコール、ポリウレタンフ
ォーム、ポリスチレンフォーム、ポリアクリルアミド、
ポリビニルフォルマール樹脂多孔質体、シリコンフォー
ム、セルロース多孔質体等の発泡体あるいは樹脂が好ま
しい。酵母の増殖および活性が低下したあるいは死滅し
た酵母の脱離等を考慮すると、多孔質の担体が好まし
い。多孔質体の開口部の大きさは細胞によっても異なる
が、酵母が十分に入り込めて、増殖できる大きさが適当
であり、５０μｍ〜１、０００μｍが好適であるが、こ
れに限定されない。(Immobilized yeast having lipase on cell surface) "Carrier" means a substance capable of immobilizing yeast, and is preferably a substance which is insoluble in water or a specific solvent. . As the material of the carrier used in the present invention, for example, polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide,
A foam or resin such as a polyvinyl formal resin porous body, silicon foam, or a cellulose porous body is preferable. In consideration of the growth of yeast and the elimination of yeast having reduced or dead yeast activity, a porous carrier is preferred. The size of the opening of the porous body varies depending on the cell, but it is appropriate that the yeast can sufficiently enter and grow, and the size is suitably 50 μm to 1,000 μm, but is not limited thereto.
【００３６】また、担体の形状は問わない。担体の強
度、培養効率等を考慮すると、球状あるいは立方体状
で、大きさは、球状の場合、直径が２ｍｍ〜５０ｍｍ、
立方体状の場合、２ｍｍ〜５０ｍｍ角が好ましい。The shape of the carrier is not limited. In consideration of the strength of the carrier, cultivation efficiency, etc., it is spherical or cubic, and the size is 2 mm to 50 mm in the case of spherical,
In the case of a cubic shape, 2 mm to 50 mm square is preferable.
【００３７】「固定化」とは、酵母が遊離の状態ではな
い状態を意味し、例えば、酵母が担体に結合あるいは付
着または担体内部に取り込まれた状態等をいう。酵母の
固定化には、例えば、担体結合法、架橋法および包括法
等の公知の方法が適用できる。なかでも、凝集性の酵母
の固定化には、担体結合法が最適である。担体結合法に
は、イオン交換性の樹脂に吸着させる化学的吸着法ある
いは物理的吸着法が含まれる。The term "immobilized" means a state in which the yeast is not in a free state, for example, a state in which the yeast is bound to or adhered to a carrier or incorporated into the carrier. For immobilization of yeast, for example, known methods such as a carrier binding method, a cross-linking method and an entrapping method can be applied. Above all, the carrier binding method is most suitable for immobilizing cohesive yeast. The carrier binding method includes a chemical adsorption method or a physical adsorption method in which the carrier is adsorbed on an ion exchange resin.
【００３８】本発明のリパーゼを細胞表層に有する凝集
性酵母は、担体に固定化されているにもかかわらず、増
殖可能であり、そして活性が低下すると自然に脱落して
いく性質を有しているため、担体に結合した酵母は、生
菌数がほぼ一定に保たれ、活性が高いという特徴があ
る。この特徴を考慮すると、担体への結合は物理的吸着
が最も好ましい。物理的吸着には特別な手段は必要な
い。凝集性あるいは接着性の細胞と前記多孔質の担体と
を単に混合して培養することにより、細胞が多孔質体の
開口部に入りこみ、担体に付着する。The agglutinating yeast having the lipase of the present invention on the cell surface is capable of proliferating despite being immobilized on a carrier, and has a property of dropping off naturally when its activity decreases. Therefore, the yeast bound to the carrier is characterized in that the viable cell count is kept almost constant and the activity is high. Considering this feature, physical adsorption is most preferred for binding to the carrier. No special measures are required for physical adsorption. By simply mixing and culturing the cohesive or adhesive cells and the porous carrier, the cells enter the opening of the porous body and adhere to the carrier.
【００３９】「凝集性」とは、液体中に浮遊または分散
して存在する酵母等が、集合して塊（集合体）を作る性
質を意味し、「接着性」とは、酵母同士が接着または結
合し、集合体を形成する性質を意味する。The term "cohesiveness" refers to the property of yeasts or the like floating or dispersed in a liquid to form a lump (aggregate), and the term "adhesiveness" refers to the property that yeasts adhere to each other. Or, it means the property of binding to form an aggregate.
【００４０】「活性が低下した」とは、酵母自体は死滅
していないものの細胞全体の活性が弱まった状態、ある
いは、たとえば凝集に関する活性が低下する、凝集に関
する酵素をコードするＤＮＡのレベルで活性が弱まる等
の状態となり、凝集できなくなる状態をいう。The term “reduced activity” refers to a state in which the yeast itself has not been killed but the activity of the whole cell has been weakened, or the activity is at the level of a DNA encoding an enzyme relating to aggregation, for example, the activity relating to aggregation is reduced. Is a state in which the particles cannot be aggregated.
【００４１】また、本発明においては、凝集性または接
着性の酵母は、凝集または接着に関する遺伝子の導入に
より凝集性または接着性を付与された酵母であってもよ
い。In the present invention, the agglutinating or adhesive yeast may be a yeast to which the aggregating or adhesive property is imparted by introducing a gene relating to agglutination or adhesion.
【００４２】「凝集または接着に関する遺伝子」とは、
凝集または接着に関与する物質、例えば、酵母における
キチン、レクチン等をコードする構造遺伝子は、もちろ
ん、凝集性に関する遺伝子としては、ＦＬＯ１(J.Watar
iら、Agric.Biol.Chem., 55,1547(1991), G.G.Stewart
ら、Can.J. Microbiol., 23, 441(1977), I.Russell
ら、Inst.Brew., 86, 120(1980), C.W.Lewisら、J.Ins
t.Brew., 82 158(1976))等、ＦＬＯ５(I.Russellら、J.
Inst.Brew., 85, 95(1979))、およびＦＬＯ８(I.Yamash
itaら、Agric.Biol.Chem., 48 131(1984))等の遺伝子が
挙げられる。"A gene relating to aggregation or adhesion"
Structural genes encoding substances involved in aggregation or adhesion, for example, chitin, lectin and the like in yeast, of course, include FLO1 (J.
i et al., Agric. Biol. Chem., 55, 1547 (1991), GGStewart.
Et al., Can.J.Microbiol., 23, 441 (1977), I. Russell.
Inst. Brew., 86, 120 (1980), CWLewis et al., J. Ins.
t.Brew., 82 158 (1976)), FLO5 (I. Russell et al., J.
Inst. Brew., 85, 95 (1979)), and FLO8 (I.
Gene et al., Agric. Biol. Chem., 48 131 (1984)).
【００４３】これらの凝集または接着に関する遺伝子
は、上記の出発材料のプラスミドに組み込まれて、本発
明のＤＮＡとともに酵母に導入される。These genes relating to aggregation or adhesion are incorporated into the above-mentioned plasmid of the starting material, and introduced into yeast together with the DNA of the present invention.
【００４４】このようにして得られる固定化された酵母
は、担体に付着した状態で、浮遊状態で培養されるか、
カラム等に充填されて、いわゆるバイオリアクターとし
て用いることができる。連続的にあるいはバッチで繰り
返し培養しても、活性が低下したあるいは死滅した細胞
が脱離していくので、活性が落ちることはなく、有効に
利用することができる。The immobilized yeast thus obtained may be cultured in a suspended state while attached to a carrier, or
It can be packed in a column or the like and used as a so-called bioreactor. Even if the cells are continuously or batch-cultured, the cells whose activity has decreased or have died out are eliminated, so that the activity does not decrease and the cells can be effectively used.
【００４５】（脂肪酸および脂肪酸エステルの製造方
法）本発明の、リパーゼを細胞表層に有する酵母と油脂
とを反応させて、脂肪酸および脂肪酸エステルを製造す
る方法を説明する。(Method for Producing Fatty Acids and Fatty Acid Esters) A method for producing fatty acids and fatty acid esters by reacting a yeast having a lipase on the cell surface with fats and oils according to the present invention will be described.
【００４６】油脂としては、植物、動物油（魚、哺乳動
物等）などに含まれるトリアシルグリセロールが好適に
用いられる。また、これらを用いる食用油脂、その廃油
なども好適に用いられる。As the fats and oils, triacylglycerols contained in plant and animal oils (fish, mammals, etc.) are preferably used. Edible fats and oils using these, waste oils thereof, and the like are also suitably used.
【００４７】本発明に用いられる油脂としては、例え
ば、サラダ油、ごま油、ヤシ油、パーム油、落花生油、
菜種油、ラー油、ベニバナ油、綿実油、カカオ脂、オリ
ーブ油、大豆油、トール油、ひまし油等の植物油脂、牛
脂、豚脂、鯨油、魚油（いわし油）等の動物油脂、およ
びこれらの廃油が挙げられるが、これらに限定されな
い。The oils and fats used in the present invention include, for example, salad oil, sesame oil, coconut oil, palm oil, peanut oil,
Vegetable oils and fats such as rapeseed oil, chili oil, safflower oil, cottonseed oil, cocoa butter, olive oil, soybean oil, tall oil, castor oil, animal fats and oils such as tallow, lard, whale oil, fish oil (sardine oil), and waste oils thereof However, it is not limited to these.
【００４８】特に、廃油は、廃棄物としての処理が難し
く、処理方法が環境問題となっている。本発明によれ
ば、廃油の処理が容易に行え、かつ燃料としてリサイク
ルされる上、極めて安価であることからコストの点で有
利である等の、環境、技術およびコストの面でその効果
は大きなものである。In particular, it is difficult to treat waste oil as waste, and the treatment method is an environmental problem. ADVANTAGE OF THE INVENTION According to this invention, the effect is large in terms of environment, technology, and cost such that waste oil can be easily treated and recycled as fuel, and it is extremely inexpensive and advantageous in terms of cost. Things.
【００４９】リパーゼを細胞表層に有する酵母と油脂と
の反応は、公知の方法が用いられる。脂肪酸の製造の場
合には、基質である油脂の水分に対する濃度は、用いる
リパーゼの種類により異なるが、一般には、約５重量％
（以下、単に％）〜８０％が好ましく、約１０％〜３０
％がさらに好ましい。反応は、油脂を溶解する有機溶媒
の存在下でも行うことができる。温度は、約２０℃〜６
０℃が好ましい。耐熱性のリパーゼの場合は、さらに高
温で行うことができる。この場合、酵母が生育できなく
ても、表層のリパーゼは活性を有したまま固定されてい
るので、使用できる。A known method is used for the reaction between the yeast having the lipase on the cell surface and the oil or fat. In the case of producing fatty acids, the concentration of the fat or oil as a substrate with respect to the moisture varies depending on the type of lipase to be used, but generally about 5% by weight.
(Hereinafter simply referred to as "%") to 80%, preferably about 10% to 30%.
% Is more preferred. The reaction can be performed even in the presence of an organic solvent that dissolves fats and oils. The temperature is about 20 ° C-6
0 ° C. is preferred. In the case of a thermostable lipase, it can be performed at a higher temperature. In this case, even if the yeast cannot grow, the lipase on the surface layer can be used because it is fixed while maintaining its activity.
【００５０】反応のｐＨは、リパーゼの性質を考慮して
決定されるが、約４．０〜８．０が好ましい。反応の進
行とともにｐＨは低下するので、ｐＨを一定の値あるい
は特定の範囲に調節することが好ましい。The pH of the reaction is determined in consideration of the properties of the lipase, but is preferably about 4.0 to 8.0. Since the pH decreases as the reaction proceeds, it is preferable to adjust the pH to a certain value or a specific range.
【００５１】脂肪酸エステルは、リパーゼと油脂との反
応を、アルコールの存在下で行うことにより、得られ
る。アルコールとしては、メタノール、エタノール、プ
ロパノールなどの低級アルコールが好ましい。アルコー
ルは、油脂のモル数の１倍ないしそれ以上加えるとよ
い。この反応により、脂肪酸エステルが得られ、この脂
肪酸エステルは、そのまま、燃料（いわゆるバイオディ
ーゼル燃料）として用いることができる。The fatty acid ester can be obtained by reacting lipase with fats and oils in the presence of alcohol. As the alcohol, lower alcohols such as methanol, ethanol and propanol are preferable. It is preferable that the alcohol is added in an amount of one or more times the number of moles of the fat or oil. By this reaction, a fatty acid ester is obtained, and this fatty acid ester can be used as it is as a fuel (so-called biodiesel fuel).
【００５２】リパーゼと油脂との反応は、固定化され
た、リパーゼを細胞表層に有する酵母をカラムにつめ、
あるいは、攪拌槽にいれて、連続的、半回分的あるいは
回分的に行われる。反応の経時変化を、例えば、ガスク
ロマトグラフやＨＰＬＣなどでモニターして、基質の添
加、反応時間などを決定すれば良い。The reaction between the lipase and the fat or oil is carried out by packing the immobilized yeast having the lipase on the cell surface in a column,
Alternatively, it is carried out continuously, semi-batchwise or batchwise in a stirring tank. The time-dependent change of the reaction may be monitored by, for example, gas chromatography or HPLC to determine the addition of the substrate, the reaction time, and the like.
【００５３】反応終了後、脂肪酸あるいは脂肪酸エステ
ルとグリセロールやモノグリセリド、ジグリセリドと
は、例えば遠心分離器による分離操作および蒸留操作や
晶析操作等により分離され、脂肪酸エステルが単離され
る。After completion of the reaction, the fatty acid or fatty acid ester is separated from glycerol, monoglyceride, and diglyceride by, for example, a separation operation using a centrifuge, a distillation operation, a crystallization operation, or the like, and the fatty acid ester is isolated.
【００５４】得られる脂肪酸またはそのエステルは、用
いる油脂にもよるが、例えば、カプリル酸、２−エチル
ヘキシル酸、ペラルゴン酸、ラウリン酸、ミリスチン
酸、ミリストレイン酸、パルミチン酸、イソパルミチン
酸、パルミトレイン酸、マルガリン酸、ステアリン酸、
イソステアリン酸、ペトロセリン酸、オレイン酸、リノ
ール酸、α-リノレン酸、γ-リノレン酸、アラキン酸、
ゴンドイン酸、ベヘン酸、セトレイン酸、エルカ酸、あ
るいはこれらのエステル、またはこれらの混合物が挙げ
られる。The resulting fatty acid or ester thereof depends on the fat or oil to be used. For example, caprylic acid, 2-ethylhexylic acid, pelargonic acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, isopalmitic acid, palmitoleic acid , Margaric acid, stearic acid,
Isostearic acid, petroselinic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachiic acid,
Gondoic acid, behenic acid, cetreic acid, erucic acid, or esters thereof, or mixtures thereof.
【００５５】（油脂のエステル交換）リパーゼを細胞表
層に有する酵母と油脂とを、脂肪酸の存在下、反応させ
ると、油脂のエステル交換反応が起こる。エステル交換
反応の条件は、水分濃度を除き、脂肪酸を製造する条件
とほぼ同じである。水分濃度はできるだけ低い方がよ
く、一般に数％以下である。この値は、用いるリパーゼ
の種類、濃度等に依存して変動する。添加する脂肪酸と
しては、上記の脂肪酸が挙げられる。好適に用いられる
脂肪酸は、用途に応じて異なるが、ステアリン酸、オレ
イン酸、リノール酸、リノレン酸等である。(Transesterification of fats and oils) When a yeast having lipase on the cell surface and fats and oils are reacted in the presence of a fatty acid, transesterification of fats and oils occurs. The conditions of the transesterification reaction are almost the same as the conditions for producing fatty acids except for the water concentration. The water concentration should be as low as possible, and is generally several percent or less. This value varies depending on the type and concentration of the lipase used. The fatty acids to be added include the above-mentioned fatty acids. Suitable fatty acids vary depending on the application, but include stearic acid, oleic acid, linoleic acid, linolenic acid and the like.
【００５６】以下、実施例を挙げて本発明を説明する
が、本発明がこの実施例により限定されるものではな
い。Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
【００５７】[0057]
【実施例】（実施例１：分泌シグナル配列、リパーゼの
構造遺伝子配列、α-アグルチニンの一部の配列及びＧ
ＰＩアンカー付着シグナル配列をこの順で有するＤＮＡ
の作成）作成の模式図を、図３に示す。EXAMPLES (Example 1: Secretory signal sequence, structural gene sequence of lipase, partial sequence of α-agglutinin and G
DNA having a PI anchor attachment signal sequence in this order
FIG. 3 shows a schematic diagram of the creation.
【００５８】Ａ：分泌シグナルを有するリパーゼ遺伝子
の取得 J.Biochem.116巻、536-540(1994)（以下、単に文献とい
う）に記載の方法に従って、Fusarium heterosporumの
リパーゼのｃDNAを取得した。簡単に述べると、まず、F
usarium heterosporumから全ＲＮＡを抽出し、つい
で、オリゴdTセルロースを用いてPoly(A)+RNAを取得
し、Okayama-Berg cDNAライブラリー合成キット（東洋
紡製）を用いて、cDNAをE.coli DH5αに導入して、ｃD
NAライブラリーを作成した。A: Acquisition of Lipase Gene Having Secretion Signal According to the method described in J. Biochem. 116, 536-540 (1994) (hereinafter simply referred to as literature), cDNA of Fusarium heterosporum lipase was obtained. Briefly, first, F
Total RNA was extracted from usarium heterosporum, then Poly (A) + RNA was obtained using oligo dT cellulose, and cDNA was transformed into E. coli DH5α using the Okayama-Berg cDNA library synthesis kit (Toyobo). Introduce, cD
NA library was created.
【００５９】5'-GCAGCATCAGCATGCTG-3’（配列番号：
１）を合成し、これをプローブとしてコロニーハイブリ
ダイゼーションを行い、ハイブリダイズするクローンを
得て、サンガー法により、配列を決定した。得られたク
ローンの制限酵素地図、およびプレプロ配列は、文献に
記載のものと一致した。得られたクローンを、PstIで部
分分解し、SpeIで切断して、約1.2kbpの長さの断片を得
た。このPstI-SpeI断片は５’上流側の非コード領域配
列、リパーゼのプレプロ配列（分泌シグナルとリパーゼ
の構造遺伝子）及び３’非コード領域配列を有してい
た。5'-GCAGCATCAGCATGCTG-3 '(SEQ ID NO:
1) was synthesized, and colony hybridization was performed using this as a probe to obtain a hybridizing clone, and the sequence was determined by the Sanger method. The restriction map of the clone obtained and the prepro sequence were consistent with those described in the literature. The resulting clone was partially digested with PstI and cut with SpeI to obtain a fragment having a length of about 1.2 kbp. This PstI-SpeI fragment had a 5 ′ upstream non-coding region sequence, a lipase prepro sequence (secretory signal and lipase structural gene) and a 3 ′ non-coding region sequence.
【００６０】Ｂ．α-アグルチニンのC末端の一部をコー
ドする配列及びＧＰＩアンカー付着シグナル配列を有す
る遺伝子の取得 酵母のα-アグルチニンの遺伝子の一部（320アミノ酸）
を有する配列を取得するため、酵母Saccharomyces cere
visiae MT8-1から、常法により染色体DNAを単離し、5'-
GTACCTCGAGCGCCAAAAGCTCTTTTATC-3'（配列番号：２）お
よび5'-GCGGTACCTTTGATTATGTTCTTTCTAT-3'（配列番号：
３）の２つのプライマーを用いて、PCRを行った。得ら
れた断片をXhoIとKpnIとで消化して、XhoI-KpnI断片を
得た。このXhoI-KpnI断片には、α-アグルチニンのＣ末
端から320アミノ酸をコードする配列と、３’非コード
領域の466bpとを含んでおり、この配列中にＧＰＩアン
カー付着シグナル配列が含まれていた。配列表の配列番
号：８にその配列を示す。配列表には記載されていない
が、TCGA配列（XhoIの切断によって生じた配列）が5'末
端に付着している。B. Acquisition of a sequence coding for a part of the C-terminal of α-agglutinin and a gene having a GPI anchor attachment signal sequence Part of a yeast α-agglutinin gene (320 amino acids)
Yeast strain Saccharomyces cere
From siae MT8-1, chromosomal DNA was isolated by a standard method and 5'-
GTACCTCGAGCGCCAAAAGCTCTTTTATC-3 ′ (SEQ ID NO: 2) and 5′-GCGGTACCTTTGATTATGTTCTTTCTAT-3 ′ (SEQ ID NO:
PCR was performed using the two primers in 3). The obtained fragment was digested with XhoI and KpnI to obtain an XhoI-KpnI fragment. This XhoI-KpnI fragment contained a sequence encoding 320 amino acids from the C-terminal of α-agglutinin and 466 bp of the 3 ′ non-coding region, and this sequence contained a GPI anchor attachment signal sequence. . The sequence is shown in SEQ ID NO: 8 in the sequence listing. Although not described in the sequence listing, a TCGA sequence (a sequence generated by cleavage of XhoI) is attached to the 5 'end.
【００６１】目的のＤＮＡは、Ａで得られた分泌シグナ
ルを有するリパーゼ遺伝子とＢで得られたXhoI-KpnI断
片を接続することにより得られる。リパーゼとアグルチ
ニンの融合タンパク質を作成するために、以下の操作を
行った。The target DNA can be obtained by connecting the lipase gene having a secretion signal obtained in A with the XhoI-KpnI fragment obtained in B. The following operation was performed to create a fusion protein of lipase and agglutinin.
【００６２】リパーゼの終止コドンをなくし、AvaIIIと
EcoRIの制限酵素部位を導入するために、以下のプライ
マー：5'-AAGCAGATGCATTTAGGCCATGGAGAGAATTCG-3'（配
列番号：４）を、そして、5'側の非コード領域にEcoRI
部位を導入するために、以下のプライマー：5'-TCTCAAT
ATGAGAGTGAATTCGGAAGGATTGTC-3'（配列番号：５）を用
いて、PCRを行った。The lipase stop codon was eliminated, and AvaIII and
To introduce EcoRI restriction enzyme sites, the following primers were used: 5'-AAGCAGATGCATTTAGGCCATGGAGAGAATTCG-3 '(SEQ ID NO: 4) and EcoRI was added to the 5' non-coding region.
To introduce the site, the following primer: 5'-TCTCAAT
PCR was performed using ATGAGAGTGAATTCGGAAGGATTGTC-3 ′ (SEQ ID NO: 5).
【００６３】PCR後EcoRIで消化し、得られた断片をpBR3
22のEcoRI部位に導入してDNAを増幅した。EcoRI切断断
片中にAvaIII部位を有するDNAは5'側と3'側の両方の変
異を含んでいる。そこで、まず、AvaIIIで切断した後、
XhoI切断部位配列を有するリンカー：5'-TGCATTACTCGAG
GG-3'(配列番号：６) 3'-AATGAGCTCCC-5'(配列番号：７)を結合し、ついで、E
coRIとXhoIとで切断して、約1.1kbpのEcoRI-XhoI断片を
取得した。After PCR, the fragment was digested with EcoRI, and the obtained fragment was digested with pBR3
DNA was amplified by introducing it into 22 EcoRI sites. DNA having an AvaIII site in the EcoRI digestion fragment contains both 5 ′ and 3 ′ mutations. So, first, after cutting with AvaIII,
Linker with XhoI cleavage site sequence: 5'-TGCATTACTCGAG
GG-3 '(SEQ ID NO: 6) 3'-AATGAGCTCCC-5' (SEQ ID NO: 7) was bound,
Cleavage was performed with coRI and XhoI to obtain an EcoRI-XhoI fragment of about 1.1 kbp.
【００６４】得られたEcoRI-XhoI断片とα-アグルチニ
ンのXhoI-KpnI断片とをプラスミドpYE22mのEcoRIとXhoI
切断部位に挿入して、目的のプラスミドpLP11を得た。The obtained EcoRI-XhoI fragment and α-agglutinin XhoI-KpnI fragment were combined with EcoRI and XhoI of plasmid pYE22m.
Insertion into the cleavage site gave the desired plasmid pLP11.
【００６５】（実施例２：細胞表層にリパーゼを有する
酵母の作成）プラスミドpLP11を単離し、酢酸リチウム
法で酵母Saccharomyces cerevisiaeHF7Cに導入した。0.
67%Yeast nitrogen base w/o amino acids、2%グルコー
スを含むＳＤ寒天培地で培養し、生育してきた酵母を選
択した。この酵母は、プラスミドpLP11を有しており、S
accharomyces cerevisiae HF7C（ｐLP11）と名付けた
（以下、単にHF7C (pLP11)という）。得られた酵母HF7C
(pLP11)をＳＤ液体培地で培養し、遠心分離して培地と
菌体とに分離し、それぞれのリパーゼ活性を測定した。
コントロールとして、Saccharomyces cerevisiae HF7C
を用いた。その結果、コントロールは、培地および菌体
にリパーゼ活性はみとめられなかった。形質転換された
酵母HF7C (pLP11)の培地中にはほとんどリパーゼ活性は
みられなかったが、酵母HF7C (pLP11)菌体はリパーゼ活
性を有していた。Example 2 Preparation of Yeast Having Lipase on Cell Surface Plasmid pLP11 was isolated and introduced into yeast Saccharomyces cerevisiae HF7C by the lithium acetate method. 0.
Yeasts grown and cultured on an SD agar medium containing 67% yeast nitrogen base w / o amino acids and 2% glucose were selected. This yeast has the plasmid pLP11 and
accharomyces cerevisiae HF7C (pLP11) (hereinafter simply referred to as HF7C (pLP11)). Yeast HF7C obtained
(pLP11) was cultured in an SD liquid medium, centrifuged to separate the medium into cells, and the lipase activity of each was measured.
As a control, Saccharomyces cerevisiae HF7C
Was used. As a result, in the control, no lipase activity was observed in the medium and the cells. Almost no lipase activity was found in the medium of the transformed yeast HF7C (pLP11), but the yeast HF7C (pLP11) cells had lipase activity.
【００６６】なお、リパーゼ活性の有無は、50mM KOH
を含むオリーブ油を30℃、30分間、500rpmで振とうし
て、遊離する脂肪酸を測定して行った。The presence or absence of lipase activity was determined by using 50 mM KOH
Was shaken at 500 rpm for 30 minutes at 30 ° C. to measure the released fatty acids.
【００６７】（実施例３：細胞表層にリパーゼを有する
酵母の固定化）ＳＤ培地３Ｌを含む気泡塔型培養槽に、
6mm角のポリウレタンフォームBSPsを、1,000個/Lとなる
ように投入して、酵母HF7C (pLP11)とともに６〜２０Ｌ
／分の通気量で培養した。これにより、ポリウレタンフ
ォームに酵母HF7C (pLP11)が固定された。Example 3 Immobilization of Yeast Having Lipase on Cell Surface In a bubble column type culture tank containing 3 L of SD medium,
6mm square polyurethane foam BSPs are charged at 1,000 cells / L, and 6-20L together with yeast HF7C (pLP11).
Per minute. As a result, yeast HF7C (pLP11) was immobilized on the polyurethane foam.
【００６８】（実施例４：固定化された細胞表層にリパ
ーゼを有する酵母による油脂の分解）実施例３で得られ
たポリウレタンフォームに固定された酵母HF7C(pLP11)
に新たに反応液：オリーブ油１００ｇ、メチルアルコー
ル３０ｇ、およびＳＤ培地３Ｌを加え、３〜２０Ｌ／分
の通気量で気泡塔型培養槽内で３０℃、２４時間接触さ
せた。反応中、KOHを用いて、ｐＨを５．０〜６．０に
コントロールした。第１サイクル目の反応液を回収した
後、新たな反応液を加え、２サイクル目の反応を行い、
これを５サイクルまで行った。第１サイクル終了後の反
応液組成を分析した結果、培養槽内に１０ｇのオレイン
酸メチルが生産されていることがわかった。(Example 4: Decomposition of fats and oils by yeast having lipase on the immobilized cell surface) Yeast HF7C (pLP11) immobilized on the polyurethane foam obtained in Example 3
, A reaction solution: 100 g of olive oil, 30 g of methyl alcohol, and 3 L of SD medium were added, and the mixture was contacted at 30 ° C. for 24 hours in a bubble column type culture tank with an aeration rate of 3 to 20 L / min. During the reaction, the pH was controlled at 5.0 to 6.0 using KOH. After collecting the reaction solution in the first cycle, a new reaction solution is added, and the reaction in the second cycle is performed.
This was performed up to 5 cycles. As a result of analyzing the composition of the reaction solution after the completion of the first cycle, it was found that 10 g of methyl oleate was produced in the culture tank.
【００６９】第２サイクル目以降も活性は落ちることな
く、固定化された酵母HF7C (pLP11)はリパーゼ活性を発
現した。固定化細胞から遊離してきた酵母を集め、活性
を測定したところ、リパーゼ活性は、大きく低下してい
た。The activity did not decrease even after the second cycle, and the immobilized yeast HF7C (pLP11) exhibited lipase activity. When the yeast released from the immobilized cells was collected and the activity was measured, the lipase activity was greatly reduced.
【００７０】[0070]
【発明の効果】細胞表層にリパーゼを有する酵母は、油
脂を脂肪酸とグリセロールに分解する。凝集性酵母を用
いる場合、簡単に固定化できると同時に、増殖可能であ
り、活性を失うと脱離するので、活性の高い酵母だけが
固定されるという利点がある。特に、廃油等からバイオ
ディーゼル燃料を製造するのに有効である。The yeast having lipase on the cell surface decomposes fats and oils into fatty acids and glycerol. When flocculant yeast is used, it can be easily immobilized, can grow at the same time, and is detached when it loses its activity, so that there is an advantage that only highly active yeast is fixed. In particular, it is effective for producing biodiesel fuel from waste oil and the like.
【００７１】[0071]
【配列表】 配列番号：１ 配列の長さ：１７ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： GCAGCATCAG CATGCTG 17 配列番号：２ 配列の長さ：２９ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： GTACCTCGAG CGCCAAAAGC TCTTTTATC 29 配列番号：３ 配列の長さ：２８ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： GCGGTACCTT TGATTATGTT CTTTCTAT 28 配列番号：４ 配列の長さ：３３ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： AAGCAGATGC ATTTAGGCCA TGGAGAGAAT TCG 33 配列番号：５ 配列の長さ：３３ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： TCTCAATATG AGAGTGAATT CGGAAGGATT GTC 33 配列番号：６ 配列の長さ：１５ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： TGCATTACTC GAGGG 15 配列番号：７ 配列の長さ：１１ 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： CCCTCGAGTA A 8 配列番号：８ 配列の長さ： 配列の型：核酸 鎖の数：１本鎖 トポロジー：直線状 配列： AGC GCC AAA AGC TCT TTT ATC TCA ACC ACT ACT ACT GAT TTA ACA AGT 48 Ser Ala Lys Ser Ser Phe Ile Ser Thr Thr Thr Thr Asp Leu Thr Ser 1 5 10 15 ATA AAC ACT AGT GCG TAT TCC ACT GGA TCC ATT TCC ACA GTA GAA ACA 96 Ile Asn Thr Ser Ala Tyr Ser Thr Gly Ser Ile Ser Thr Val Glu Thr 20 25 30 GGC AAT CGA ACT ACA TCA GAA GTG ATC AGT CAT GTG GTG ACT ACC AGC 144 Gly Asn Arg Thr Thr Ser Glu Val Ile Ser His Val Val Thr Thr Ser 35 40 45 ACA AAA CTG TCT CCA ACT GCT ACT ACC AGC CTG ACA ATT GCA CAA ACC 192 Thr Lys Leu Ser Pro Thr Ala Thr Thr Ser Leu Thr Ile Ala Gln Thr 50 55 60 AGT ATC TAT TCT ACT GAC TCA AAT ATC ACA GTA GGA ACA GAT ATT CAC 240 Ser Ile Tyr Ser Thr Asp Ser Asn Ile Thr Val Gly Thr Asp Ile His 65 70 75 80 ACC ACA TCA GAA GTG ATT AGT GAT GTG GAA ACC ATT AGC AGA GAA ACA 288 Thr Thr Ser Glu Val Ile Ser Asp Val Glu Thr Ile Ser Arg Glu Thr 85 90 95 GCT TCG ACC GTT GTA GCC GCT CCA ACC TCA ACA ACT GGA TGG ACA GGC 336 Ala Ser Thr Val Val Ala Ala Pro Thr Ser Thr Thr Gly Trp Thr Gly 100 105 110 GCT ATG AAT ACT TAC ATC TCG CAA TTT ACA TCC TCT TCT TTC GCA ACA 384 Ala Met Asn Thr Tyr Ile Ser Gln Phe Thr Ser Ser Ser Phe Ala Thr 115 120 125 ATC AAC AGC ACA CCA ATA ATC TCT TCA TCA GCA GTA TTT GAA ACC TCA 432 Ile Asn Ser Thr Pro Ile Ile Ser Ser Ser Ala Val Phe Glu Thr Ser 130 135 140 GAT GCT TCA ATT GTC AAT GTG CAC ACT GAA AAT ATC ACG AAT ACT GCT 480 Asp Ala Ser Ile Val Asn Val His Thr Glu Asn Ile Thr Asn Thr Ala 145 150 155 160 GCT GTT CCA TCT GAA GAG CCC ACT TTT GTA AAT GCC ACG AGA AAC TCC 528 Ala Val Pro Ser Glu Glu Pro Thr Phe Val Asn Ala Thr Arg Asn Ser 165 170 175 TTA AAT TCC TTC TGC AGC AGC AAA CAG CCA TCC AGT CCC TCA TCT TAT 576 Leu Asn Ser Phe Cys Ser Ser Lys Gln Pro Ser Ser Pro Ser Ser Tyr 180 185 190 ACG TCT TCC CCA CTC GTA TCG TCC CTC TCC GTA AGC AAA ACA TTA CTA 624 Thr Ser Ser Pro Leu Val Ser Ser Leu Ser Val Ser Lys Thr Leu Leu 195 200 205 AGC ACC AGT TTT ACG CCT TCT GTG CCA ACA TCT AAT ACA TAT ATC AAA 672 Ser Thr Ser Phe Thr Pro Ser Val Pro Thr Ser Asn Thr Tyr Ile Lys 210 215 220 ACG AAA AAT ACG GGT TAC TTT GAG CAC ACG GCT TTG ACA ACA TCT TCA 720 Thr Lys Asn Thr Gly Tyr Phe Glu His Thr Ala Leu Thr Thr Ser Ser 225 230 235 240 GTT GGC CTT AAT TCT TTT AGT GAA ACA GCA GTC TCA TCT CAG GGA ACG 768 Val Gly Leu Asn Ser Phe Ser Glu Thr Ala Val Ser Ser Gln Gly Thr 245 250 255 AAA ATT GAC ACC TTT TTA GTG TCA TCC TTG ATC GCA TAT CCT TCT TCT 816 Lys Ile Asp Thr Phe Leu Val Ser Ser Leu Ile Ala Tyr Pro Ser Ser 260 265 270 GCA TCA GGA AGC CAA TTG TCC GGT ATC CAA CAG AAT TTC ACA TCA ACT 864 Ala Ser Gly Ser Gln Leu Ser Gly Ile Gln Gln Asn Phe Thr Ser Thr 275 280 285 TCT CTC ATG ATT TCA ACC TAT GAA GGT AAA GCG TCT ATA TTT TTC TCA 912 Ser Leu Met Ile Ser Thr Tyr Glu Gly Lys Ala Ser Ile Phe Phe Ser 290 295 300 GCT GAG CTC GGT TCG ATC ATT TTT CTG CTT TTG TCG TAC CTG CTA TTC 960 Ala Glu Leu Gly Ser Ile Ile Phe Leu Leu Leu Ser Tyr Leu Leu Phe 305 310 315 320 TAAAACGGGT ACTGTACAGT TAGTACATTG AGTCGAAATA TACGAAATTA TTGTTCATAA 1020 TTTTCATCCT GGCTCTTTTT TTCTTCAACC ATAGTTAAAT GGACAGTTCA TATCTTAAAC 1080 TCTAATAATA CTTTTCTAGT TCTTATCCTT TTCCGTCTCA CCGCAGATTT TATCATAGTA 1140 TTAAATTTAT ATTTTGTTCG TAAAAAGAAA AATTTGTGAG CGTTACCGCT CGTTTCATTA 1200 CCCGAAGGCT GTTTCAGTAG ACCACTGATT AAGTAAGTAG ATGAAAAAAT TTCATCACCA 1260 TGAAAGAGTT CGATGAGAGC TACTTTTTCA AATGCTTAAC AGCTAACCGC CATTCAATAA 1320 TGTTACGTTC TCTTCATTCT GCGGCTACGT TATCTAACAA GAGGTTTTAC TCTCTCATAT 1380 CTCATTCAAA TAGAAAGAAC ATAATCAAA 1409[Sequence list] SEQ ID NO: 1 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: GCAGCATCAG CATGCTG 17 SEQ ID NO: 2 Sequence length: 29 Sequence type: Nucleic acid Number of strands: single strand Topology: linear Sequence: GTACCTCGAG CGCCAAAAGC TCTTTTATC 29 SEQ ID NO: 3 Sequence length: 28 Sequence type: nucleic acid Number of strands: 1 strand Topology: linear sequence: GCGGTACCTT TGATTATGTT CTTTCTAT 28 sequence No .: 4 Sequence length: 33 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence: AAGCAGATGC ATTTAGGCCA TGGAGAGAAT TCG 33 SEQ ID NO: 5 Sequence length: 33 Sequence type: Number of nucleic acid strands Single-stranded topology: linear Sequence: TCTCAATATG AGAGTGAATT CGGAAGGATT GTC 33 SEQ ID NO: 6 Sequence length: 15 Sequence type: nucleic acid Number of strands: Single-stranded topology: linear sequence: TGCATTACTC GAGGG 15 SEQ ID NO: 7 Array length: 11 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence: CCCTCGAGTA A 8 SEQ ID NO: 8 Sequence length: Sequence type: Number of nucleic acid strands: Single strand Topology: Linear sequence: AGC GCC AAA AGC TCT TTT ATC TCA ACC ACT ACT ACT GAT TTA ACA AGT 48 Ser Ala Lys Ser Ser Phe Ile Ser Thr Thr Thr Thr Asp Leu Thr Ser 1 5 10 15 ATA AAC ACT AGT GCG TAT TCC ACT GGA TCC ATT TCC ACA GTA GAA ACA 96 Ile Asn Thr Ser Ala Tyr Ser Thr Gly Ser Ile Ser Thr Val Glu Thr 20 25 30 GGC AAT CGA ACT ACA TCA GAA GTG ATC AGT CAT GTG GTG ACT ACC AGC 144 Gly Asn Arg Thr Thr Ser Glu Val Ile Ser His Val Val Thr Thr Ser 35 40 45 ACA AAA CTG TCT CCA ACT GCT ACT ACC AGC CTG ACA ATT GCA CAA ACC 192 Thr Lys Leu Ser Pro Thr Ala Thr Thr Ser Leu Thr Ile Ala Gln Thr 50 55 60 AGT ATC TAT TCT ACT GAC TCA AAT ATC ACA GTA GGA ACA GAT ATT CAC 240 Ser Ile Tyr Ser Thr Asp Ser Asn Ile Thr Val Gly Thr Asp Ile His 65 70 75 80 ACC ACA TCA GAA GTG ATT AGT GAT GTG GAA ACC ATT AGC AGA GAA ACA 288 T hr Thr Ser Glu Val Ile Ser Asp Val Glu Thr Ile Ser Arg Glu Thr 85 90 95 GCT TCG ACC GTT GTA GCC GCT CCA ACC TCA ACA ACT GGA TGG ACA GGC 336 Ala Ser Thr Val Val Ala Ala Pro Thr Ser Thr Thr Gly Trp Thr Gly 100 105 110 GCT ATG AAT ACT TAC ATC TCG CAA TTT ACA TCC TCT TCT TTC GCA ACA 384 Ala Met Asn Thr Tyr Ile Ser Gln Phe Thr Ser Ser Ser Phe Ala Thr 115 120 125 ATC AAC AGC ACA CCA ATA ATC TCT TCA TCA GCA GTA TTT GAA ACC TCA 432 Ile Asn Ser Thr Pro Ile Ile Ser Ser Ser Ala Val Phe Glu Thr Ser 130 135 140 GAT GCT TCA ATT GTC AAT GTG CAC ACT GAA AAT ATC ACG AAT ACT GCT 480 Asp Ala Ser Ile Val Asn Val His Thr Glu Asn Ile Thr Asn Thr Ala 145 150 155 160 GCT GTT CCA TCT GAA GAG CCC ACT TTT GTA AAT GCC ACG AGA AAC TCC 528 Ala Val Pro Ser Glu Glu Pro Thr Phe Val Asn Ala Thr Arg Asn Ser 165 170 175 TTA AAT TCC TTC TGC AGC AGC AAA CAG CCA TCC AGT CCC TCA TCT TAT 576 Leu Asn Ser Phe Cys Ser Ser Lys Gln Pro Ser Ser Pro Ser Ser Tyr 180 185 190 ACG TCT TCC CCA CTC GTA TCG TCC CTC TCC GTA AGC AAA ACA TTA CTA 624 Thr Ser Ser Pro Leu Val Ser Ser Leu Ser Val Ser Lys Thr Leu Leu 195 200 205 AGC ACC AGT TTT ACG CCT TCT GTG CCA ACA TCT AAT ACA TAT ATC AAA 672 Ser Thr Ser Phe Thr Pro Ser Val Pro Thr Ser Asn Thr Tyr Ile Lys 210 215 220 ACG AAA AAT ACG GGT TAC TTT GAG CAC ACG GCT TTG ACA ACA TCT TCA 720 Thr Lys Asn Thr Gly Tyr Phe Glu His Thr Ala Leu Thr Thr Ser Ser 225 230 235 240 GTT GGC CTT AAT TCT TTT AGT GAA ACA GCA GTC TCA TCT CAG GGA ACG 768 Val Gly Leu Asn Ser Phe Ser Glu Thr Ala Val Ser Ser Gln Gly Thr 245 250 255 AAA ATT GAC ACC TTT TTA GTG TCA TCC TTG ATC GCA TAT CCT TCT TCT 816 Lys Ile Asp Thr Phe Leu Val Ser Ser Leu Ile Ala Tyr Pro Ser Ser 260 265 270 GCA TCA GGA AGC CAA TTG TCC GGT ATC CAA CAG AAT TTC ACA TCA ACT 864 Ala Ser Gly Ser Gln Leu Ser Gly Ile Gln Gln Asn Phe Thr Ser Thr 275 280 285 TCT CTC ATG ATT TCA ACC TAT GAA GGT AAA GCG TCT ATA TTT TTC TCA 912 Ser Leu Met Ile Ser Thr Tyr Glu Gly Lys Ala Ser Ile Phe Phe Ser 290 295 300 GCT GAG CTC GGT TCG ATC ATT TTT CTG CTT TTG TCGTAC CTG CTA TTC 960 Ala Glu Leu Gly Ser Ile Ile Phe Leu Leu Leu Ser Tyr Leu Leu Phe 305 310 315 320 TAAAACGGGT ACTGTACAGT TAGTACATTG AGTCGAAATA TACGAAATTA TTGTTCATAA 1020 TTTTCATCCT GGCTCTTTTT TTCTTCAACC ATAGTTAAAT GGACAGTTCA TATCTTAAAC 1080 TCTAATAATA CTTTTCTAGT TCTTATCCTT TTCCGTCTCA CCGCAGATTT TATCATAGTA 1140 TTAAATTTAT ATTTTGTTCG TAAAAAGAAA AATTTGTGAG CGTTACCGCT CGTTTCATTA 1200 CCCGAAGGCT GTTTCAGTAG ACCACTGATT AAGTAAGTAG ATGAAAAAAT TTCATCACCA 1260 TGAAAGAGTT CGATGAGAGC TACTTTTTCA AATGCTTAAC AGCTAACCGC CATTCAATAA 1320 TGTTACGTTC TCTTCGATCTATCTACGATCTACTATCTCGATCTATCTCAGCTAATC
【図１】本発明のＤＮＡの構造を示す模式図である。FIG. 1 is a schematic diagram showing the structure of the DNA of the present invention.
【図２】細胞表層局在タンパク質の輸送システムを示す
模式図である。FIG. 2 is a schematic diagram showing a transport system for a protein localized on a cell surface layer.
【図３】プラスミドpLP11の作成の模式図である。FIG. 3 is a schematic diagram showing the construction of plasmid pLP11.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ //(Ｃ１２Ｎ 15/09 ＺＮＡ Ｃ１２Ｒ 1:865） (Ｃ１２Ｎ 9/18 Ｃ１２Ｒ 1:77） (Ｃ１２Ｎ 11/08 Ｃ１２Ｒ 1:865） (Ｃ１２Ｐ 7/62 Ｃ１２Ｒ 1:865） ──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 6 Identification symbol FI // (C12N 15/09 ZNA C12R 1: 865) (C12N 9/18 C12R 1:77) (C12N 11/08 C12R 1: 865) ) (C12P 7/62 C12R 1: 865)
Claims (11)
子配列、細胞表層局在タンパク質の一部をコードする配
列及びＧＰＩアンカー付着シグナル配列をこの順で有す
るＤＮＡであって、リパーゼを細胞表層に発現し得るＤ
ＮＡ。1. A DNA comprising, in this order, a secretory signal sequence, a lipase structural gene sequence, a sequence encoding a part of a cell surface localization protein, and a GPI anchor attachment signal sequence, wherein the lipase is expressed on the cell surface. Get D
NA.
ードする配列及びアンカー付着シグナル配列が酵母のα
-アグルチニンの配列である、請求項１に記載のＤＮ
Ａ。2. A sequence encoding a part of the cell surface localization protein and an anchor attachment signal sequence are those of yeast α.
2. The DN of claim 1, which is a sequence of -agglutinin.
A.
ードする配列及びアンカー付着シグナル配列を有する配
列が、α−アグルチニンのＣ末端から３２０アミノ酸の
配列である、請求項２に記載のＤＮＡ。3. The DNA according to claim 2, wherein the sequence encoding a part of the cell surface localization protein and the sequence having an anchor attachment signal sequence are a sequence of 320 amino acids from the C-terminal of α-agglutinin.
由来のリパーゼである、請求項１ないし３いずれかの項
に記載のＤＮＡ。4. The method according to claim 1, wherein the lipase is Fusarium heterosporum.
The DNA according to any one of claims 1 to 3, wherein the DNA is a lipase derived from.
請求項１ないし４いずれかの項に記載のＤＮＡ。5. The method according to claim 5, wherein the DNA is in the form of a plasmid.
The DNA according to any one of claims 1 to 4.
て、請求項１ないし５いずれかの項に記載のＤＮＡを有
する、酵母。6. A yeast having a lipase on a cell surface, the yeast having the DNA according to any one of claims 1 to 5.
請求項６に記載の酵母。7. The yeast, which is immobilized on a carrier,
The yeast according to claim 6.
ある、請求項６または７に記載の酵母。8. The yeast according to claim 6, wherein the yeast is a cohesive or adhesive yeast.
酵母と油脂とを反応させる工程を含む、脂肪酸の製造方
法。9. A method for producing a fatty acid, comprising a step of reacting the yeast according to claim 6 with an oil or fat.
８いずれかの項に記載の酵母と油脂とを反応させる工程
を含む、脂肪酸エステルの製造方法。10. A method for producing a fatty acid ester, comprising a step of reacting the yeast according to any one of claims 6 to 8 with an oil or fat in the presence of an alcohol.
の酵母と油脂とを、必要に応じて脂肪酸の存在下、反応
させる工程を含む、油脂のエステル交換方法。11. A method for transesterifying an oil or fat, comprising a step of reacting the yeast according to claim 6 with an oil or fat in the presence of a fatty acid, if necessary.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10097937A JPH11290078A (en) | 1998-04-09 | 1998-04-09 | Yeast having lipase on cell surface and its utilization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10097937A JPH11290078A (en) | 1998-04-09 | 1998-04-09 | Yeast having lipase on cell surface and its utilization |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11290078A true JPH11290078A (en) | 1999-10-26 |
Family
ID=14205590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10097937A Pending JPH11290078A (en) | 1998-04-09 | 1998-04-09 | Yeast having lipase on cell surface and its utilization |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11290078A (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001038553A1 (en) * | 1999-11-26 | 2001-05-31 | Kansai Chemical Engineering Co., Ltd. | Process for producing fatty acid lower alcohol ester |
| WO2002085935A1 (en) * | 2001-04-19 | 2002-10-31 | Kansai Chemical Engineering Co., Ltd. | Cell surface layer-binding protein and utilization thereof |
| JPWO2002042483A1 (en) * | 2000-11-27 | 2004-03-25 | 関西化学機械製作株式会社 | Process for producing ethanol from starch |
| KR100462832B1 (en) * | 2002-04-11 | 2004-12-20 | 한국과학기술원 | Gene for Cell-surface Expression |
| KR100475133B1 (en) * | 2002-09-13 | 2005-03-10 | 한국생명공학연구원 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| JP2008092849A (en) * | 2006-10-11 | 2008-04-24 | Kobe Univ | Highly active arming yeast having highly active lipase b in cell surface layer, method for producing the same and highly active lipase b |
| WO2009128305A1 (en) | 2008-04-14 | 2009-10-22 | 三井化学株式会社 | Yeast for production of glucose, and method for production of glucose using the same |
| USRE43135E1 (en) | 2001-05-18 | 2012-01-24 | Danisco A/S | Method of improving dough and bread quality |
| USRE43341E1 (en) | 1995-06-07 | 2012-05-01 | Danisco A/S | Method of improving the properties of a flour dough, a flour dough improving composition and improved food products |
| US8252577B2 (en) | 2008-04-30 | 2012-08-28 | Bio-Energy Corporation | Microorganism that displays biotin on cell surface |
| US8889371B2 (en) | 2004-07-16 | 2014-11-18 | Dupont Nutrition Biosciences Aps | Lipolytic enzyme: uses thereof in the food industry |
| US9580729B2 (en) | 2012-03-26 | 2017-02-28 | Kansai Chemical Engineering Co., Ltd. | Method for producing ethanol |
| US9816113B2 (en) | 2013-09-04 | 2017-11-14 | Kansai Chemical Engineering Co., Ltd. | Method for producing ethanol |
| JP2018170988A (en) * | 2017-03-31 | 2018-11-08 | シーシーアイホールディングス株式会社 | Method for producing viable bacterial preparation, and viable bacterial preparation, and wastewater treatment method using the same |
| US11306300B2 (en) | 2017-05-11 | 2022-04-19 | Kansai Chemical Engineering Co., Ltd. | Microorganism capable of displaying α-galactosidase on surface layer thereof, and use thereof |
-
1998
- 1998-04-09 JP JP10097937A patent/JPH11290078A/en active Pending
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE43341E1 (en) | 1995-06-07 | 2012-05-01 | Danisco A/S | Method of improving the properties of a flour dough, a flour dough improving composition and improved food products |
| WO2001038553A1 (en) * | 1999-11-26 | 2001-05-31 | Kansai Chemical Engineering Co., Ltd. | Process for producing fatty acid lower alcohol ester |
| US6982155B1 (en) | 1999-11-26 | 2006-01-03 | Kansai Chemical Engineering Co., Ltd. | Process for producing fatty acid lower alcohol ester |
| JP4666884B2 (en) * | 2000-11-27 | 2011-04-06 | 関西化学機械製作株式会社 | Method for producing ethanol from starch |
| JPWO2002042483A1 (en) * | 2000-11-27 | 2004-03-25 | 関西化学機械製作株式会社 | Process for producing ethanol from starch |
| WO2002085935A1 (en) * | 2001-04-19 | 2002-10-31 | Kansai Chemical Engineering Co., Ltd. | Cell surface layer-binding protein and utilization thereof |
| US7192764B2 (en) | 2001-04-19 | 2007-03-20 | Kansai Chemical Engineering, Co. | Cell surface layer-binding protein and utilization thereof |
| KR100714670B1 (en) * | 2001-04-19 | 2007-05-07 | 간사이가가쿠기카이세이사쿠가부시키가이샤 | Cell surface layer-binding protein and utilization thereof |
| USRE43135E1 (en) | 2001-05-18 | 2012-01-24 | Danisco A/S | Method of improving dough and bread quality |
| KR100462832B1 (en) * | 2002-04-11 | 2004-12-20 | 한국과학기술원 | Gene for Cell-surface Expression |
| KR100475133B1 (en) * | 2002-09-13 | 2005-03-10 | 한국생명공학연구원 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| US8889371B2 (en) | 2004-07-16 | 2014-11-18 | Dupont Nutrition Biosciences Aps | Lipolytic enzyme: uses thereof in the food industry |
| JP2008092849A (en) * | 2006-10-11 | 2008-04-24 | Kobe Univ | Highly active arming yeast having highly active lipase b in cell surface layer, method for producing the same and highly active lipase b |
| WO2009128305A1 (en) | 2008-04-14 | 2009-10-22 | 三井化学株式会社 | Yeast for production of glucose, and method for production of glucose using the same |
| US8252577B2 (en) | 2008-04-30 | 2012-08-28 | Bio-Energy Corporation | Microorganism that displays biotin on cell surface |
| US9580729B2 (en) | 2012-03-26 | 2017-02-28 | Kansai Chemical Engineering Co., Ltd. | Method for producing ethanol |
| US9816113B2 (en) | 2013-09-04 | 2017-11-14 | Kansai Chemical Engineering Co., Ltd. | Method for producing ethanol |
| JP2018170988A (en) * | 2017-03-31 | 2018-11-08 | シーシーアイホールディングス株式会社 | Method for producing viable bacterial preparation, and viable bacterial preparation, and wastewater treatment method using the same |
| US11306300B2 (en) | 2017-05-11 | 2022-04-19 | Kansai Chemical Engineering Co., Ltd. | Microorganism capable of displaying α-galactosidase on surface layer thereof, and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JPH11290078A (en) | Yeast having lipase on cell surface and its utilization | |
| Takahashi et al. | Extracellular production of active Rhizopus oryzae lipase by Saccharomyces cerevisiae | |
| US11603545B2 (en) | Recombinant yeast strain for producing nervonic acids and application thereof | |
| EP1042451A1 (en) | Linoleate isomerase | |
| CN107475268A (en) | From the lipase gene and its Related product of trichoderma and application | |
| US20160222361A1 (en) | Myrmecia incisa reisigl diacylglycerol acyltransferase gene sequence and use thereof | |
| JP5438259B2 (en) | Yeast presenting Candida antarctica-derived lipase B on the cell surface | |
| CN108251400B (en) | Lipase and application thereof | |
| KR100714670B1 (en) | Cell surface layer-binding protein and utilization thereof | |
| JP2021522845A (en) | Mutant lipase and its use | |
| US20050186583A1 (en) | Linoleate isomerase | |
| CN108251401A (en) | Lipase and its application | |
| AU2016320187A1 (en) | Lipid production method | |
| CN106676083B (en) | Lipase extraction method | |
| Lock et al. | Lipases produced by yeasts: powerful biocatalysts for industrial purposes | |
| Simatupang et al. | Lipolytic activity of Itb1. 1 and Lk3 thermostable lipases expressed in Escherichia coli and Pichia pastoris | |
| JP4836106B2 (en) | Method for producing optically active substance using cell surface display enzyme | |
| CN108330114B (en) | EPA-utilizing diacylglycerol acyltransferase and application thereof | |
| US20230250406A1 (en) | Mutant lipase and use thereof | |
| KR101897189B1 (en) | Lipase derived from Penicillium chrysogenum and preparation method of lipase using the same | |
| JP2007020539A (en) | Arabinofuranosidase b-presenting yeast and utilization thereof | |
| JP2005245335A (en) | Target protein-expressing yeast and use thereof | |
| KR20160042254A (en) | Process for preparing lipase derived from Candida antarctica using Pichia pastoris | |
| JP2005245334A (en) | Arabinofuranosidase-expressing yeast and use thereof | |
| CN111206023A (en) | Metabolic engineering method for efficiently improving content of microalgae triglyceride |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040601 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20040802 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20050705 |