CN100529069C - 脂解酶变体 - Google Patents
脂解酶变体 Download PDFInfo
- Publication number
- CN100529069C CN100529069C CNB028035852A CN02803585A CN100529069C CN 100529069 C CN100529069 C CN 100529069C CN B028035852 A CNB028035852 A CN B028035852A CN 02803585 A CN02803585 A CN 02803585A CN 100529069 C CN100529069 C CN 100529069C
- Authority
- CN
- China
- Prior art keywords
- variant
- lipolytic enzyme
- seq
- parental generation
- ester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 67
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 67
- 230000002366 lipolytic effect Effects 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 35
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 10
- 229920001131 Pulp (paper) Polymers 0.000 claims abstract description 9
- 102220598657 5-hydroxytryptamine receptor 1E_D27R_mutation Human genes 0.000 claims description 96
- 150000002148 esters Chemical class 0.000 claims description 17
- 238000012216 screening Methods 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 102220487741 Protein eyes shut homolog_D27N_mutation Human genes 0.000 claims description 11
- 235000001014 amino acid Nutrition 0.000 claims description 11
- 241000233866 Fungi Species 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 102200161847 rs104894126 Human genes 0.000 claims description 10
- 102220056652 rs397514616 Human genes 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 9
- 102200027496 rs63751429 Human genes 0.000 claims description 9
- 235000015927 pasta Nutrition 0.000 claims description 8
- 102220010822 rs397514616 Human genes 0.000 claims description 8
- 239000004744 fabric Substances 0.000 claims description 7
- 102200121669 rs104894915 Human genes 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 102220209560 rs1057521321 Human genes 0.000 claims description 4
- 102200034886 rs121912642 Human genes 0.000 claims description 4
- 102200034317 rs28938169 Human genes 0.000 claims description 4
- 102220085035 rs552361923 Human genes 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 102220571370 FUN14 domain-containing protein 1_N25L_mutation Human genes 0.000 claims description 2
- 102220539527 Prostaglandin D2 receptor 2_S58Y_mutation Human genes 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 102220197780 rs121434596 Human genes 0.000 claims description 2
- 102200025824 rs3743171 Human genes 0.000 claims description 2
- 102220257095 rs587779102 Human genes 0.000 claims description 2
- 102220056648 rs730881059 Human genes 0.000 claims description 2
- 102200015540 rs75295839 Human genes 0.000 claims description 2
- 102220084740 rs767734253 Human genes 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 238000004925 denaturation Methods 0.000 claims 2
- 230000036425 denaturation Effects 0.000 claims 2
- 102220052839 rs73113102 Human genes 0.000 claims 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 230000028327 secretion Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000002538 fungal effect Effects 0.000 abstract 1
- 108090001060 Lipase Proteins 0.000 description 46
- 102000004882 Lipase Human genes 0.000 description 40
- 239000004367 Lipase Substances 0.000 description 38
- 235000019421 lipase Nutrition 0.000 description 38
- 108020004414 DNA Proteins 0.000 description 21
- 241000223258 Thermomyces lanuginosus Species 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 18
- 241000228212 Aspergillus Species 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 102100031415 Hepatic triacylglycerol lipase Human genes 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 7
- 238000013016 damping Methods 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 238000000113 differential scanning calorimetry Methods 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000228245 Aspergillus niger Species 0.000 description 5
- 241000235403 Rhizomucor miehei Species 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 240000006439 Aspergillus oryzae Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000223221 Fusarium oxysporum Species 0.000 description 4
- 239000006142 Luria-Bertani Agar Substances 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 102220021416 rs80357110 Human genes 0.000 description 4
- 108010061238 threonyl-glycine Proteins 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 101001065065 Aspergillus awamori Feruloyl esterase A Proteins 0.000 description 3
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 3
- 102220565484 Killer cell immunoglobulin-like receptor 2DL2_D96E_mutation Human genes 0.000 description 3
- 108020002496 Lysophospholipase Proteins 0.000 description 3
- 244000271379 Penicillium camembertii Species 0.000 description 3
- 235000002245 Penicillium camembertii Nutrition 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 3
- 235000019626 lipase activity Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002708 random mutagenesis Methods 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 108010011667 Ala-Phe-Ala Proteins 0.000 description 2
- XRUJOVRWNMBAAA-NHCYSSNCSA-N Ala-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 XRUJOVRWNMBAAA-NHCYSSNCSA-N 0.000 description 2
- 241000351920 Aspergillus nidulans Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 2
- 241000144128 Lichtheimia corymbifera Species 0.000 description 2
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 2
- 102100037611 Lysophospholipase Human genes 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102100033357 Pancreatic lipase-related protein 2 Human genes 0.000 description 2
- 240000005384 Rhizopus oryzae Species 0.000 description 2
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 2
- QGVBFDIREUUSHX-IFFSRLJSSA-N Thr-Val-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O QGVBFDIREUUSHX-IFFSRLJSSA-N 0.000 description 2
- 101150050575 URA3 gene Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960003311 ampicillin trihydrate Drugs 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 229920002301 cellulose acetate Polymers 0.000 description 2
- 238000000978 circular dichroism spectroscopy Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000010985 leather Substances 0.000 description 2
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 101150116440 pyrF gene Proteins 0.000 description 2
- 101150054232 pyrG gene Proteins 0.000 description 2
- 102220245301 rs1555596673 Human genes 0.000 description 2
- 102200142660 rs7565275 Human genes 0.000 description 2
- 102220087418 rs864622444 Human genes 0.000 description 2
- NNRFRJQMBSBXGO-CIUDSAMLSA-N (3s)-3-[[2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NNRFRJQMBSBXGO-CIUDSAMLSA-N 0.000 description 1
- QOPBEBWGSGFROG-UHFFFAOYSA-N 2-(1h-indol-2-yl)acetic acid Chemical class C1=CC=C2NC(CC(=O)O)=CC2=C1 QOPBEBWGSGFROG-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 241001375492 Absidia reflexa Species 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 1
- KMGOBAQSCKTBGD-DLOVCJGASA-N Ala-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CN=CN1 KMGOBAQSCKTBGD-DLOVCJGASA-N 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 1
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 1
- LSMDIAAALJJLRO-XQXXSGGOSA-N Ala-Thr-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LSMDIAAALJJLRO-XQXXSGGOSA-N 0.000 description 1
- JJHBEVZAZXZREW-LFSVMHDDSA-N Ala-Thr-Phe Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O JJHBEVZAZXZREW-LFSVMHDDSA-N 0.000 description 1
- LTTLSZVJTDSACD-OWLDWWDNSA-N Ala-Thr-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O LTTLSZVJTDSACD-OWLDWWDNSA-N 0.000 description 1
- KLKARCOHVHLAJP-UWJYBYFXSA-N Ala-Tyr-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CS)C(O)=O KLKARCOHVHLAJP-UWJYBYFXSA-N 0.000 description 1
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 1
- KHOITXIGCFIULA-UHFFFAOYSA-N Alophen Chemical compound C1=CC(OC(=O)C)=CC=C1C(C=1N=CC=CC=1)C1=CC=C(OC(C)=O)C=C1 KHOITXIGCFIULA-UHFFFAOYSA-N 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 1
- JSHVMZANPXCDTL-GMOBBJLQSA-N Arg-Asp-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JSHVMZANPXCDTL-GMOBBJLQSA-N 0.000 description 1
- OBFTYSPXDRROQO-SRVKXCTJSA-N Arg-Gln-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCN=C(N)N OBFTYSPXDRROQO-SRVKXCTJSA-N 0.000 description 1
- SKTGPBFTMNLIHQ-KKUMJFAQSA-N Arg-Glu-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SKTGPBFTMNLIHQ-KKUMJFAQSA-N 0.000 description 1
- WVNFNPGXYADPPO-BQBZGAKWSA-N Arg-Gly-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O WVNFNPGXYADPPO-BQBZGAKWSA-N 0.000 description 1
- URAUIUGLHBRPMF-NAKRPEOUSA-N Arg-Ser-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O URAUIUGLHBRPMF-NAKRPEOUSA-N 0.000 description 1
- BRCVLJZIIFBSPF-ZLUOBGJFSA-N Asn-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N BRCVLJZIIFBSPF-ZLUOBGJFSA-N 0.000 description 1
- RCENDENBBJFJHZ-ACZMJKKPSA-N Asn-Asn-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O RCENDENBBJFJHZ-ACZMJKKPSA-N 0.000 description 1
- KXEGPPNPXOKKHK-ZLUOBGJFSA-N Asn-Asp-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O KXEGPPNPXOKKHK-ZLUOBGJFSA-N 0.000 description 1
- IBLAOXSULLECQZ-IUKAMOBKSA-N Asn-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC(N)=O IBLAOXSULLECQZ-IUKAMOBKSA-N 0.000 description 1
- MYTHOBCLNIOFBL-SRVKXCTJSA-N Asn-Ser-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MYTHOBCLNIOFBL-SRVKXCTJSA-N 0.000 description 1
- DPSUVAPLRQDWAO-YDHLFZDLSA-N Asn-Tyr-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)N)N DPSUVAPLRQDWAO-YDHLFZDLSA-N 0.000 description 1
- WJHYGGVCWREQMO-GHCJXIJMSA-N Asp-Cys-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WJHYGGVCWREQMO-GHCJXIJMSA-N 0.000 description 1
- KYQNAIMCTRZLNP-QSFUFRPTSA-N Asp-Ile-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O KYQNAIMCTRZLNP-QSFUFRPTSA-N 0.000 description 1
- XYPJXLLXNSAWHZ-SRVKXCTJSA-N Asp-Ser-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XYPJXLLXNSAWHZ-SRVKXCTJSA-N 0.000 description 1
- UTLCRGFJFSZWAW-OLHMAJIHSA-N Asp-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UTLCRGFJFSZWAW-OLHMAJIHSA-N 0.000 description 1
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 1
- SFJUYBCDQBAYAJ-YDHLFZDLSA-N Asp-Val-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SFJUYBCDQBAYAJ-YDHLFZDLSA-N 0.000 description 1
- 241000892910 Aspergillus foetidus Species 0.000 description 1
- 241000228232 Aspergillus tubingensis Species 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 108010051152 Carboxylesterase Proteins 0.000 description 1
- 102000013392 Carboxylesterase Human genes 0.000 description 1
- 241000122205 Chamaeleonidae Species 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- BYALSSDCQYHKMY-XGEHTFHBSA-N Cys-Arg-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)N)O BYALSSDCQYHKMY-XGEHTFHBSA-N 0.000 description 1
- KVCJEMHFLGVINV-ZLUOBGJFSA-N Cys-Ser-Asn Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(N)=O KVCJEMHFLGVINV-ZLUOBGJFSA-N 0.000 description 1
- ALNKNYKSZPSLBD-ZDLURKLDSA-N Cys-Thr-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O ALNKNYKSZPSLBD-ZDLURKLDSA-N 0.000 description 1
- 241001136487 Eurotium Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000146406 Fusarium heterosporum Species 0.000 description 1
- PHZYLYASFWHLHJ-FXQIFTODSA-N Gln-Asn-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PHZYLYASFWHLHJ-FXQIFTODSA-N 0.000 description 1
- XEYMBRRKIFYQMF-GUBZILKMSA-N Gln-Asp-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O XEYMBRRKIFYQMF-GUBZILKMSA-N 0.000 description 1
- JUUNNOLZGVYCJT-JYJNAYRXSA-N Gln-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JUUNNOLZGVYCJT-JYJNAYRXSA-N 0.000 description 1
- JTWZNMUVQWWGOX-SOUVJXGZSA-N Gln-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCC(=O)N)N)C(=O)O JTWZNMUVQWWGOX-SOUVJXGZSA-N 0.000 description 1
- BUVMZWZNWMKASN-QEJZJMRPSA-N Glu-Asn-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)N)C(O)=O)=CNC2=C1 BUVMZWZNWMKASN-QEJZJMRPSA-N 0.000 description 1
- RDPOETHPAQEGDP-ACZMJKKPSA-N Glu-Asp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RDPOETHPAQEGDP-ACZMJKKPSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- OQXDUSZKISQQSS-GUBZILKMSA-N Glu-Lys-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OQXDUSZKISQQSS-GUBZILKMSA-N 0.000 description 1
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 1
- QNJNPKSWAHPYGI-JYJNAYRXSA-N Glu-Phe-Leu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=CC=C1 QNJNPKSWAHPYGI-JYJNAYRXSA-N 0.000 description 1
- QLNKFGTZOBVMCS-JBACZVJFSA-N Glu-Tyr-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QLNKFGTZOBVMCS-JBACZVJFSA-N 0.000 description 1
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 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
- GGEJHJIXRBTJPD-BYPYZUCNSA-N Gly-Asn-Gly Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GGEJHJIXRBTJPD-BYPYZUCNSA-N 0.000 description 1
- LXXLEUBUOMCAMR-NKWVEPMBSA-N Gly-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)CN)C(=O)O LXXLEUBUOMCAMR-NKWVEPMBSA-N 0.000 description 1
- TZOVVRJYUDETQG-RCOVLWMOSA-N Gly-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CN TZOVVRJYUDETQG-RCOVLWMOSA-N 0.000 description 1
- YZACQYVWLCQWBT-BQBZGAKWSA-N Gly-Cys-Arg Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YZACQYVWLCQWBT-BQBZGAKWSA-N 0.000 description 1
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 1
- XMPXVJIDADUOQB-RCOVLWMOSA-N Gly-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C([O-])=O)NC(=O)CNC(=O)C[NH3+] XMPXVJIDADUOQB-RCOVLWMOSA-N 0.000 description 1
- VAXIVIPMCTYSHI-YUMQZZPRSA-N Gly-His-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN VAXIVIPMCTYSHI-YUMQZZPRSA-N 0.000 description 1
- LPCKHUXOGVNZRS-YUMQZZPRSA-N Gly-His-Ser Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O LPCKHUXOGVNZRS-YUMQZZPRSA-N 0.000 description 1
- YIFUFYZELCMPJP-YUMQZZPRSA-N Gly-Leu-Cys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O YIFUFYZELCMPJP-YUMQZZPRSA-N 0.000 description 1
- LRQXRHGQEVWGPV-NHCYSSNCSA-N Gly-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN LRQXRHGQEVWGPV-NHCYSSNCSA-N 0.000 description 1
- BXICSAQLIHFDDL-YUMQZZPRSA-N Gly-Lys-Asn Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BXICSAQLIHFDDL-YUMQZZPRSA-N 0.000 description 1
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 1
- ZKJZBRHRWKLVSJ-ZDLURKLDSA-N Gly-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)O ZKJZBRHRWKLVSJ-ZDLURKLDSA-N 0.000 description 1
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 1
- DNAZKGFYFRGZIH-QWRGUYRKSA-N Gly-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 DNAZKGFYFRGZIH-QWRGUYRKSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 1
- LDTJBEOANMQRJE-CIUDSAMLSA-N His-Cys-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O)N LDTJBEOANMQRJE-CIUDSAMLSA-N 0.000 description 1
- LVXFNTIIGOQBMD-SRVKXCTJSA-N His-Leu-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O LVXFNTIIGOQBMD-SRVKXCTJSA-N 0.000 description 1
- BZAQOPHNBFOOJS-DCAQKATOSA-N His-Pro-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O BZAQOPHNBFOOJS-DCAQKATOSA-N 0.000 description 1
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 1
- HZMLFETXHFHGBB-UGYAYLCHSA-N Ile-Asn-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HZMLFETXHFHGBB-UGYAYLCHSA-N 0.000 description 1
- SCHZQZPYHBWYEQ-PEFMBERDSA-N Ile-Asn-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SCHZQZPYHBWYEQ-PEFMBERDSA-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
- RPZFUIQVAPZLRH-GHCJXIJMSA-N Ile-Asp-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)O)N RPZFUIQVAPZLRH-GHCJXIJMSA-N 0.000 description 1
- DURWCDDDAWVPOP-JBDRJPRFSA-N Ile-Cys-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N DURWCDDDAWVPOP-JBDRJPRFSA-N 0.000 description 1
- SLQVFYWBGNNOTK-BYULHYEWSA-N Ile-Gly-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N SLQVFYWBGNNOTK-BYULHYEWSA-N 0.000 description 1
- CKRFDMPBSWYOBT-PPCPHDFISA-N Ile-Lys-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CKRFDMPBSWYOBT-PPCPHDFISA-N 0.000 description 1
- KCTIFOCXAIUQQK-QXEWZRGKSA-N Ile-Pro-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O KCTIFOCXAIUQQK-QXEWZRGKSA-N 0.000 description 1
- XOZOSAUOGRPCES-STECZYCISA-N Ile-Pro-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XOZOSAUOGRPCES-STECZYCISA-N 0.000 description 1
- REXAUQBGSGDEJY-IGISWZIWSA-N Ile-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N REXAUQBGSGDEJY-IGISWZIWSA-N 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- MDVZJYGNAGLPGJ-KKUMJFAQSA-N Leu-Asn-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MDVZJYGNAGLPGJ-KKUMJFAQSA-N 0.000 description 1
- ZURHXHNAEJJRNU-CIUDSAMLSA-N Leu-Asp-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ZURHXHNAEJJRNU-CIUDSAMLSA-N 0.000 description 1
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 1
- NRFGTHFONZYFNY-MGHWNKPDSA-N Leu-Ile-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NRFGTHFONZYFNY-MGHWNKPDSA-N 0.000 description 1
- JKSIBWITFMQTOA-XUXIUFHCSA-N Leu-Ile-Val 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)C)C(O)=O JKSIBWITFMQTOA-XUXIUFHCSA-N 0.000 description 1
- IFMPDNRWZZEZSL-SRVKXCTJSA-N Leu-Leu-Cys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O IFMPDNRWZZEZSL-SRVKXCTJSA-N 0.000 description 1
- HVHRPWQEQHIQJF-AVGNSLFASA-N Leu-Lys-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HVHRPWQEQHIQJF-AVGNSLFASA-N 0.000 description 1
- FKQPWMZLIIATBA-AJNGGQMLSA-N Leu-Lys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FKQPWMZLIIATBA-AJNGGQMLSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- IWMJFLJQHIDZQW-KKUMJFAQSA-N Leu-Ser-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IWMJFLJQHIDZQW-KKUMJFAQSA-N 0.000 description 1
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 1
- HGLKOTPFWOMPOB-MEYUZBJRSA-N Leu-Thr-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HGLKOTPFWOMPOB-MEYUZBJRSA-N 0.000 description 1
- VHTIZYYHIUHMCA-JYJNAYRXSA-N Leu-Tyr-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O VHTIZYYHIUHMCA-JYJNAYRXSA-N 0.000 description 1
- VUBIPAHVHMZHCM-KKUMJFAQSA-N Leu-Tyr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 VUBIPAHVHMZHCM-KKUMJFAQSA-N 0.000 description 1
- 241001459306 Lichtheimia blakesleeana Species 0.000 description 1
- PBLLTSKBTAHDNA-KBPBESRZSA-N Lys-Gly-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PBLLTSKBTAHDNA-KBPBESRZSA-N 0.000 description 1
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 description 1
- BWECSLVQIWEMSC-IHRRRGAJSA-N Lys-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCCN)N BWECSLVQIWEMSC-IHRRRGAJSA-N 0.000 description 1
- YGNUDKAPJARTEM-GUBZILKMSA-N Met-Val-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O YGNUDKAPJARTEM-GUBZILKMSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- AJOKKVTWEMXZHC-DRZSPHRISA-N Phe-Ala-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 AJOKKVTWEMXZHC-DRZSPHRISA-N 0.000 description 1
- MRNRMSDVVSKPGM-AVGNSLFASA-N Phe-Asn-Gln Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MRNRMSDVVSKPGM-AVGNSLFASA-N 0.000 description 1
- MECSIDWUTYRHRJ-KKUMJFAQSA-N Phe-Asn-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O MECSIDWUTYRHRJ-KKUMJFAQSA-N 0.000 description 1
- MPFGIYLYWUCSJG-AVGNSLFASA-N Phe-Glu-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 MPFGIYLYWUCSJG-AVGNSLFASA-N 0.000 description 1
- METZZBCMDXHFMK-BZSNNMDCSA-N Phe-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N METZZBCMDXHFMK-BZSNNMDCSA-N 0.000 description 1
- KNYPNEYICHHLQL-ACRUOGEOSA-N Phe-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 KNYPNEYICHHLQL-ACRUOGEOSA-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
- 102100035200 Phospholipase A and acyltransferase 4 Human genes 0.000 description 1
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 1
- ORPZXBQTEHINPB-SRVKXCTJSA-N Pro-Arg-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1)C(O)=O ORPZXBQTEHINPB-SRVKXCTJSA-N 0.000 description 1
- XWYXZPHPYKRYPA-GMOBBJLQSA-N Pro-Asn-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XWYXZPHPYKRYPA-GMOBBJLQSA-N 0.000 description 1
- GDXZRWYXJSGWIV-GMOBBJLQSA-N Pro-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 GDXZRWYXJSGWIV-GMOBBJLQSA-N 0.000 description 1
- QGOZJLYCGRYYRW-KKUMJFAQSA-N Pro-Glu-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QGOZJLYCGRYYRW-KKUMJFAQSA-N 0.000 description 1
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 1
- SPLBRAKYXGOFSO-UNQGMJICSA-N Pro-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@@H]2CCCN2)O SPLBRAKYXGOFSO-UNQGMJICSA-N 0.000 description 1
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 description 1
- KBUAPZAZPWNYSW-SRVKXCTJSA-N Pro-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KBUAPZAZPWNYSW-SRVKXCTJSA-N 0.000 description 1
- 241001632422 Radiola linoides Species 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- FIDMVVBUOCMMJG-CIUDSAMLSA-N Ser-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO FIDMVVBUOCMMJG-CIUDSAMLSA-N 0.000 description 1
- BGOWRLSWJCVYAQ-CIUDSAMLSA-N Ser-Asp-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BGOWRLSWJCVYAQ-CIUDSAMLSA-N 0.000 description 1
- ZOHGLPQGEHSLPD-FXQIFTODSA-N Ser-Gln-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZOHGLPQGEHSLPD-FXQIFTODSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 1
- BKZYBLLIBOBOOW-GHCJXIJMSA-N Ser-Ile-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O BKZYBLLIBOBOOW-GHCJXIJMSA-N 0.000 description 1
- DOSZISJPMCYEHT-NAKRPEOUSA-N Ser-Ile-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O DOSZISJPMCYEHT-NAKRPEOUSA-N 0.000 description 1
- BSXKBOUZDAZXHE-CIUDSAMLSA-N Ser-Pro-Glu Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O BSXKBOUZDAZXHE-CIUDSAMLSA-N 0.000 description 1
- WMZVVNLPHFSUPA-BPUTZDHNSA-N Ser-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 WMZVVNLPHFSUPA-BPUTZDHNSA-N 0.000 description 1
- FHXGMDRKJHKLKW-QWRGUYRKSA-N Ser-Tyr-Gly Chemical compound OC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 FHXGMDRKJHKLKW-QWRGUYRKSA-N 0.000 description 1
- HKHCTNFKZXAMIF-KKUMJFAQSA-N Ser-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=C(O)C=C1 HKHCTNFKZXAMIF-KKUMJFAQSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 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
- JTEICXDKGWKRRV-HJGDQZAQSA-N Thr-Asn-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O JTEICXDKGWKRRV-HJGDQZAQSA-N 0.000 description 1
- MFEBUIFJVPNZLO-OLHMAJIHSA-N Thr-Asp-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O MFEBUIFJVPNZLO-OLHMAJIHSA-N 0.000 description 1
- UHBPFYOQQPFKQR-JHEQGTHGSA-N Thr-Gln-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O UHBPFYOQQPFKQR-JHEQGTHGSA-N 0.000 description 1
- IVDFVBVIVLJJHR-LKXGYXEUSA-N Thr-Ser-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IVDFVBVIVLJJHR-LKXGYXEUSA-N 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- ILDJYIDXESUBOE-HSCHXYMDSA-N Trp-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N ILDJYIDXESUBOE-HSCHXYMDSA-N 0.000 description 1
- GSCPHMSPGQSZJT-JYBASQMISA-N Trp-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O GSCPHMSPGQSZJT-JYBASQMISA-N 0.000 description 1
- UIRVSEPRMWDVEW-RNXOBYDBSA-N Trp-Tyr-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)N UIRVSEPRMWDVEW-RNXOBYDBSA-N 0.000 description 1
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-N 0.000 description 1
- NLMXVDDEQFKQQU-CFMVVWHZSA-N Tyr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NLMXVDDEQFKQQU-CFMVVWHZSA-N 0.000 description 1
- OKDNSNWJEXAMSU-IRXDYDNUSA-N Tyr-Phe-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C1=CC=C(O)C=C1 OKDNSNWJEXAMSU-IRXDYDNUSA-N 0.000 description 1
- KLQPIEVIKOQRAW-IZPVPAKOSA-N Tyr-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](CC1=CC=C(C=C1)O)N)O KLQPIEVIKOQRAW-IZPVPAKOSA-N 0.000 description 1
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 1
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 1
- ZMDCGGKHRKNWKD-LAEOZQHASA-N Val-Asn-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZMDCGGKHRKNWKD-LAEOZQHASA-N 0.000 description 1
- IRLYZKKNBFPQBW-XGEHTFHBSA-N Val-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N)O IRLYZKKNBFPQBW-XGEHTFHBSA-N 0.000 description 1
- VNGKMNPAENRGDC-JYJNAYRXSA-N Val-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=CC=C1 VNGKMNPAENRGDC-JYJNAYRXSA-N 0.000 description 1
- YKNOJPJWNVHORX-UNQGMJICSA-N Val-Phe-Thr Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YKNOJPJWNVHORX-UNQGMJICSA-N 0.000 description 1
- VSCIANXXVZOYOC-AVGNSLFASA-N Val-Pro-His Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N VSCIANXXVZOYOC-AVGNSLFASA-N 0.000 description 1
- QSPOLEBZTMESFY-SRVKXCTJSA-N Val-Pro-Val Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O QSPOLEBZTMESFY-SRVKXCTJSA-N 0.000 description 1
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 1
- HWNYVQMOLCYHEA-IHRRRGAJSA-N Val-Ser-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N HWNYVQMOLCYHEA-IHRRRGAJSA-N 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 108010058834 acylcarnitine hydrolase Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010089975 arginyl-glycyl-aspartyl-serine Proteins 0.000 description 1
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 150000001733 carboxylic acid esters Chemical group 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 238000005213 imbibition Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 108010078274 isoleucylvaline Proteins 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- -1 p-nitrophenyl ester Chemical class 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 108010093296 prolyl-prolyl-alanine Proteins 0.000 description 1
- 230000003134 recirculating effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 102220267412 rs1316316435 Human genes 0.000 description 1
- 102220100163 rs201567345 Human genes 0.000 description 1
- 102200116704 rs77587352 Human genes 0.000 description 1
- 102220093762 rs876661269 Human genes 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000007669 thermal treatment Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06L—DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
- D06L1/00—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods
- D06L1/12—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods using aqueous solvents
- D06L1/14—De-sizing
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H21/00—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties
- D21H21/02—Agents for preventing deposition on the paper mill equipment, e.g. pitch or slime control
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Textile Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
通过取代真菌脂解酶中某些特定的氨基酸残基获得具有改进的热稳定性的脂解酶变体。热稳定脂解酶变体可用于例如在制备机械纸浆或使用机械纸浆的造纸方法中控制树脂问题。
Description
技术领域
本发明涉及真菌脂解酶变体，尤其是具有改进热稳定性的变体，以及制备和使用该变体的方法。
背景技术
已知应用真菌脂解酶例如源于Thermomyces lanuginosus(同义词：Humicola lanuginosa)的脂解酶，可以用于各种工业目的，例如改进洗涤剂的效果和在纸浆和纸制造中消除树脂(pitch)问题。在某些情况下，具有改进热稳定性的脂解酶是人们所需要的(EP 374700，WO 9213130)。
WO 92/05249，WO 92/19726和WO 97/07202公开了T.lanuginosus(H.lanuginosa)脂酶的变体。
发明内容
发明人已经发现真菌脂解酶的热稳定性可通过氨基酸序列中的某些特定取代而得以改进。
因此，本发明提供一种亲代真菌脂解酶变体，该变体包括一个或多个特定氨基酸残基的取代并且其比亲代脂解酶的热稳定性高。本发明还提供一种制备脂解酶变体的方法包括：
a)筛选亲代真菌脂解酶，
b)在亲代脂解酶中取代至少一个特定的氨基酸残基，
c)可选地，取代一个或多个不同于b)中的氨基酸，
d)制备由步骤a)-c)而得的变体，
e)检测该变体的热稳定性，
f)筛选具有热稳定性升高的变体，和
g)制备所筛选的变体。
特定的氨基酸残基包括相应于SEQ ID NO：1中21，27，29，32，34-42，51，54，76，84，90-97，101，105，111，118，125，131，135，137，162，187，189，206-212，216，224-234，242-252和256的任何氨基酸残基。
热稳定性可具体升高4以上。可以用不同的氨基酸残基尤其是一个不同于Pro的氨基酸残基进行取代。
具体实施方式
亲代脂解酶
根据酶命名法(可从http://www.chem.qmw.ac.uk/iubmb/enzyme得到)本发明所用的脂解酶分类为EC 3.1.1羧酸酯水解酶。底物的特异性可以包括诸如EC 3.1.1.3三酰甘油脂酶，EC 3.1.1.4磷脂酶A2，EC 3.1.1.5溶血磷脂酶，EC 3.1.1.26半乳糖脂酶(galactolipase)，EC 3.1.1.32磷脂酶A1，EC3.1.1.73阿魏酸酯酶的活性。
亲代脂解酶是真菌的并且具有可与SEQ ID NO：1进行序列对比的氨基酸序列，SEQ ID NO：1是显示于US 5,869,438中的SEQ ID NO：2的1-269位氨基酸序列，该序列是源于Thermomyces lanuginosus(同义词：Humicolalanuginosa)的脂酶的序列，这种脂酶在EP 258 068和EP 305 216中有描述。亲代脂酶可特别与SEQ ID NO：1具有至少50％的氨基酸序列同源性。除了T.lanuginosus的脂酶外，其它的实例是沙门氏柏干酪青霉(Penicilliumcamembertii)(P25234)的脂酶，尖孢镰孢(Fusarium oxysporum)的脂酶/磷脂酶(EP 130064，WO 98/26057)，异孢镰孢(F.heterosporum)的脂酶(R87979)，臭曲霉(Aspergillus foetidus)的溶血磷脂酶(W33009)，米曲霉(A.oryzae)的磷脂酶A 1(JP-A 10-155493)，米曲霉脂酶(D85895)，黑曲霉(A.niger)的脂酶/阿魏酸酯酶(Y09330)，塔宾曲霉(A.tubingensis)脂酶/阿魏酸酯酶(Y09331)，塔宾曲霉脂酶(WO 98/45453)，黑曲霉溶血磷脂酶(WO 98/31790)，具有等电点为6.9和表观分子量为30kDa茄病镰孢(F.solanii)的脂酶(WO 96/18729)。
其它的实例是接合菌(Zygomycetes)的脂酶家族，包括具有与SEQ IDNO：2中所示序列的米赫根毛霉(Rhizomucor miehei)(P19515)的脂酶有至少50％同源性的脂酶。该家族还包括反射犁头霉(Absidia reflexa)，A.sporophora，伞枝犁头霉(A.corymbifera)，A.blakesleeana，A.griseola(在WO 96/13578和WO 97/27276中描述)和米根霉(Rhizopus oryzae)(P21811)的脂酶。括号中的数字表示公开或登录入EMBL，GenBank，GeneSeqp或Swiss-Prot数据库。
氨基酸取代
本发明的脂解酶变体包括在上述任何区的氨基酸残基的一个或多个取代。取代可以是例如在相应于SEQ ID NO：1的206-208，224-228，227-228，227-231，242-243和245-252的任何区中进行。要取代的氨基酸残基可相应于SEQ ID NO：1的Y21，D27，P29，T32，A40，F51，S54，I76，R84，I90，G91，N94，N101，S105，D111，R118，R125，A131，H135，D137，N162，V187，T189，E210，G212，S216，G225，L227，I238或P256残基。一些特定的有目的取代是那些相应于SEQ ID NO：1的D27N/R/S，P29S，T32S，F51I/L，I76V，R84C，I90L/V，G91A/N/S/T/W，L93F，N94K/R/S，F95I，D96G/N，N101D，D111A/G，R118M，A131V，H135Y，D137N，N162R，V187I，F211Y，S216P，S224I/Y，G225P，T226N，L227F/P/G/V，L227X，V228C/I，238V和P256T。
在上述区域的取代的总数通常是不高于10个，例如1，2，3，4，5，6，7或8个所述的取代。此外，本发明脂解酶变体可以任选地包括亲代酶的其它修饰，通常是不超过10个，例如不超过5个所述的修饰。与亲代脂解酶相比，变体可以特定地具有总数不超过10个的氨基酸修饰(尤其是取代)。变体一般与亲代脂解酶具有至少80％的同源性，例如至少85％，典型的至少90％或至少95％。
脂解酶变体
变体具有脂解酶活性，即其能够水解羧酸酯键以释放羧酸基(EC 3.1.1)。它可以特定地具有脂酶活性(三酰甘油脂酶活性，EC 3.1.1.3)，即在三酰甘油中水解羧酸酯键的活性，例如1，3-特异性活性。
特异性变体
下面是一些T.lanuginosus脂酶的变体的实例。相应的取代可通过在其它真菌脂解酶中相应的氨基酸取代进行：
| D27N |
| D111G+S216P |
| L227F |
| L227F+V228I |
| G225P |
| S224I+G225W+T226N+L227P+V228C |
| S224Y+G225W+T226N+L227P+V228C |
| D27R+D111G+S216P |
| D27S+D111G+S216P |
| D27N+D111A |
| D27R+D111G+S216P+L227P+P256T |
| D27R+D111G+S216P+L227G+P256T |
| D27R+D111G+S216P+L227F+P256T |
| D27R+D111G+S216P+L227V+P256T |
| D27R+D111G+S216P+L227G |
| D27R+D111G+S216P+L227X |
| D27P+D111G+S216P+L227X |
热稳定性
本申请使用合适的缓冲液在相关的PH下测定热稳定性。缓冲液和PH的实例是：pH 10.0(50mM氨基乙酸缓冲液)，pH 7.0(50mM HEPES缓冲液)或pH 5.0(50mM乙酸钠为缓冲液)。
为了比较，测定应该在相同的缓冲液，相同的条件和相同的蛋白质浓度下进行。各种方法都可用于测定热稳定性：
差示扫描量热法(Differential Scanning Calorimetry(DSC))
在DSC中，加热速度可以是每小时90度。可将样品纯化至同质(homogeneity)，熔解温度(TM)可用来表示热稳定性。
残留的酶活性
可选地，热稳定性可通过在选定的温度下温育后测定残留的脂解酶活性而得以测定。如Giver等，Proc.Natl.Acad.Sci.USA 95(1998)12809-12813和Moore等.Nat.Biotech.14(1996)458-467所述，10mMTris-HCI中对硝基苯酯，pH 7.5可用作底物。可以将样品定期地加入，或只使用一种样品，加有或没有不同的添加剂以防止或促进变性，例如在96孔中。
CD分光术
例如如Yamaguchi等Protein engineering 9(1996)789-795中所述的CD分光术。通常酶浓度约为1mg/ml，温度在5-80度之间。
变体的用途
脂解酶变体可应用于各种方法中，下面描述一些具体用途。变体通常在60-95℃(尤其是75-90℃，70-90℃或70-85℃)以及pH 4.5-11(特别是4.5-8或5-6.5)下使用。
在纸和纸浆工业中的应用
脂酶可用在制备机械纸浆或使用机械纸浆造纸的方法中避免树脂问题，其包括向纸浆中添加脂酶并温育。脂酶可以添加在所谓的回水(whitewater)(再循环工艺用水)中。它还可以用于从用过的纸上除去墨迹。通常优选的是在工业中，改进的热稳定性能够允许变体高温下使用。这可以类似于WO 9213130，WO 9207138，JP 2160984A，EP 374700进行。
在基于谷类的食品中的应用
脂解酶变体可以添加到生面团中，这种生面团可用于制备焙烤产品(特别是面包)，意大利面制品或面条。变体的改进的热稳定性允许其在加热步骤(烘焙，煮沸或油炸)期间较长时间地保持活性。这与WO 94/04035，WO 00/32758，PCT/DK 01/00472，EP 1057415类似。
添加变体可导致改进生面团的稳定性，即焙烤产品的较大块体积和/或焙烤期间尤其是加压系统(stressed system)中，例如在过分醒发成形(over-proofing)或过分揉混(over-mixing)情况中更好地保持形状。它还导致焙烤产品较低的初始硬度和/或更均一细微的碎屑(crumb)，改进的碎屑结构(较细的碎屑，较薄的孔壁(cell wall)，更圆的孔)，并且进一步改进生面团的特性例如降低生面团的柔软性，提高弹性，降低伸展性(extensibility)。
在脂肪和油工业中的用途
脂解酶变体在有机合成例如在酯的水解，合成或酯交换的方法中可用作催化剂，包括在脂解酶变体存在下酯与水的反应，酸与乙醇的反应或酯与酸，醇或第二种酯的酯交换。优选地，改进的热稳定性允许所述反应在相对高的温度下进行，相对高的温度有利于提高反应的速度和处理高熔点的底物。
上述酯可以是羧酸酯，例如甘油三酸酯。酯交换可以在存在或不存在溶剂的情况下进行。酶可以以固定化形式使用。该方法可以与WO8802775，US 6156548，US 5776741，EP 792106，EP 93602或EP 307154类似进行。
在纺织工业中的用途
变体可以用于从织物中酶去除疏水性酯的方法中，该方法包括用有效量的脂解酶处理织物以从织物中去除疏水性酯。处理可以在75℃或高于75℃的温度下进行例如1-24小时。处理之前先用脂酶变体的水溶液浸透织物至吸液比(liquor pick-up ratio)为50-200％，并后接洗涤和漂清以除去脂肪酸。
该方法与US 5578489或US 6077316中的类似。
在洗涤剂中的用途
变体可用做洗涤剂添加剂，例如浓度为(表示为纯酶蛋白)0.001-10(例如0.01-1)mg/g洗涤剂或0.001-100(例如0.01-10)mg/升洗液。这可与WO 97/04079，WO 97/07202，WO 97/41212，WO 98/08939和WO 97/43375类似进行。
皮革中的用途
类似于GB 2233665或EP 505920，本发明的变体还可用于皮革工业中。
氨基酸取代的命名
本文用于定义氨基酸取代的命名法使用的是如WO 92/05249中所述的单字母代码。
因此，D27N表示的是在位置27用N取代D。D27N/R表示用N或R取代D27。L227X表示用任一氨基酸取代L227。D27N+D111A表示两种取代的组合。
同源性和序列对比
为了实现本发明的目的，同源性的程度可以通过本领域已知的计算机程序测定，例如GCG程序包(Program Manual for the Wisconsin Package，Version 8，August 1994，Genetics Computer Group，575 Science Drive，Madison，Wisconsin，USA 53711)(Needleman，S.B.and Wunsch，C.D.，(1970)，Journal of Molecular Biology，48，443-45)中提供的GAP，使用GAP和下列设置用于多肽序列的比较：GAP生成罚分(creation penalty)3.0和GAP延伸罚分(extension penalty)0.1。
在本发明中，米赫根毛霉(rhimi)，德列马根霉(rhidl)，Thermomyceslanuginosa(former；Humicola lanuginosa)(SP400)，沙门氏柏干酪青霉(Pcl)和尖孢镰孢(Fusarium oxysporum)(FoLnp11)脂酶序列中相应的(或同源)位置通过与WO 00/32758的图1所示序列对比确定。
为了找到在序列对比中未显示的脂酶序列的同源性位置，将目的序列与图1中所示的序列进行对比。将新序列通过使用GAP序列对比与由GAP程序发现的最高同源性序列于图1中排列进行对比。GCG程序包中提供GAP(Program Manual for the Wisconsin Package，Version 8，August 1994，Genetics Computer Group，575 Science Drive，Madison，Wisconsin，USA 53711)(Needleman，S.B.and Wunsch，C.D.，(1970)，Journal of Molecular Biology，48，443-45)。使用下列设置进行多肽序列的比较：GAP生成罚分3.0和GAP延伸罚分0.1。
获得热稳定变体的方法
脂解酶的变体可以通过本领域已知的方法获得，例如描述于WO9522615或WO 0032758中的例如定点突变，随机突变或定位突变。
给定的亲代脂解酶的热稳定变体可以通过下面标准的方法获得：
突变(易错，掺杂的寡核苷酸(doped oligo)，spiked oligo)
初次筛选
更高温度稳定突变体的鉴定
维持(甘油培养，LB-Amp板，小量制备)
在另一试验板上划线-第二次筛选(比初次筛选高1度)
DNA测序
在曲霉属菌株中转化
100ml规模的培养，纯化，DSC
初次筛选实验
下面的鉴定方法用于筛选脂解酶变体并鉴定具有改进的热稳定性的变体。
例如通过易错PCR，随机诱变或定域随机诱变或通过有利的突变体和饱和诱变组合制备携带有脂解酶变体基因的大肠杆菌细胞。
在LB琼脂平板上使用滤膜实施鉴定。大肠杆菌细胞在由LB琼脂平板提供营养成分的醋酸纤维素滤膜上并且在添加氨苄西林的LB琼脂平板选择压下生长。包括所需酶的蛋白质在位于LB琼脂和醋酸纤维素滤膜之间的硝酸纤维素滤膜上收集。硝酸纤维素滤膜在所需pH(通常6.0)的缓冲液和所需温度下温育15分钟(例如对于T.lanuginosus脂酶为78度)。在冰水中猝灭滤膜后，残留的脂酶活性如Kynclova，E等(Journal of MolecularRecognition 8(1995)139-145)所述，通过裂解吲哚乙酸盐并且随后用氮蓝四唑氯化物染色反应产物进行测定。
调节所使用的热处理使亲代具有轻微活性，即与室温温育样品相比，约5-10％的活性。这有利于鉴定有用的突变体。
实施例
实施例1：脂酶的表达
质粒pMT2188
米曲霉表达质粒pCaHj 483(WO 98/00529)由基于黑曲霉中性淀粉酶II启动子与构巢曲霉丙糖磷酸异构酶非翻译前导序列(Pna2/tpi)和黑曲霉淀粉葡糖苷酶终止子(amyloglycosidase terminater(Tamg))融合的表达盒构成。质粒中还有来自构巢曲霉的曲霉选择性标记amdS，其能在以乙酰胺为唯一氮源时生长。将这些元件克隆入大肠杆菌载体pUC19(New EnglandBiolabs)。能在大肠杆菌中进行筛选的该质粒的氨苄西林抗性标记被酿酒糖酵母的URA3标记置换，这可弥补大肠杆菌中pyrF突变，置换以下面的方式进行：
pUC19复制起点是以引物142779(SEQ ID NO：3)和142780(SEQ IDNO：4)进行自pCaHj483的PCR扩增。
引物142780在PCR片段中引入Bbul位点。按照制造商对此和随后PCR的扩增的说明使用Expand PCR系统(Roche Molecular Biochemicals，Basel，Switserland)扩增。
URA3基因自通常的酿酒糖酵母克隆载体pYES2(Invitrogencorporation，Carlsbad，Ca，USA)使用引物140288(SEQ ID 5)和142778(SEQID 6)扩增。
引物140288在PCR片段中引入EcoRl位点。两种PCR片段通过将其混合并且使用引物142780和140288扩增通过重叠的方法(overlap method)在剪接中融合(Horton et al(1989)Gene，77，61-68)。
所得的片段用EcoRl和Bbul消化并且与用相同酶消化的pCaHj 483的最大片段连接。连接混合物用于转化通过Mandel和Higa方法(Mandel，M.and A.Higa(1970)J.Mol.Biol.45，154)制备的感受态pyrF大肠杆菌菌株DB6507(ATCC 35673)。转化体在添加有1g/l酪蛋白氨基酸，500μg/l硫胺素和10mg/l卡那霉素的固体M9培养基上筛选(Sambrook et.al(1989)Molecular cloning，a laboratory manual，2.edition，Cold Spring HarborLaboratory Press)。
来自所选转化体的质粒称为pCaHj 527。将存在于pCaHj527的Pna2/tpi启动子通过单PCR方法进行定点突变。
使用突变引物141223(SEQ ID NO：9)将核苷酸134-144从SEQ IDNO：7改变为SEQ ID NO：8。
使用突变引物141222(SEQ ID 12)将核苷酸423-436从SEQ ID NO：10改变为SEQ ID NO：11。
所得的质粒称为pMT2188。
质粒pENI1849
为了截短pyrG基因成pyrG表达的必需序列以减少质粒的大小，由此而改进转化频率而制备质粒pENI1849。使用pENI1299(在WO 00/24883中描述)为模板以及引物270999J8(SEQ ID 13)和270999J9(SEQ ID 14)制备PCR片段(约1800bp)。
使用限制酶StuI和SphI切割该PCR-片段，并克隆入也用StuI和SphI切割的pEN11298中(在WO 0024883中描述)；克隆通过测序证实。
质粒pENI1861
制备质粒pEN11861使在表达质粒中具有曲霉启动子表达质粒状态，以及大量的用于克隆的限制位点。
使用pMT2188(见上)作为模板以及引物051199J1(SEQ ID 15)和1298TAKA(SEQ ID 16)制备PCR片段(约620bp)。
用BssHII和Bgl II切割片段，并克隆入也用BssHII和Bgl II切割的pENI1849中。克隆通过测序证实。
质粒pENI1902
制备质粒pENI1902以使其具有能在大肠杆菌和曲霉中作用的启动子。这通过使用由推荐的“Chameleon双链定点诱变试剂盒”通过独特的位点去除实现。
质粒pENI1861用作模板以及具有5’磷酸化的下面的引物用作筛选引物：177996(SEQ ID 17)，135640(SEQ ID 18)和135638(SEQ ID 19)。
具有5’磷酸化的080399J19引物(SEQ ID NO：20)用作突变引物以在曲霉菌属表达启动子中引入-35和-10启动子共有序列(自大肠杆菌)。通过测序证实突变的引入。
质粒pSMin001
制备质粒pSMin001以允许T.lanuginosus脂酶在大肠杆菌和曲霉中表达。
使用质粒pAHL(在WO9205249中描述)为模板利用下面的引物进行PCR以扩增T.lanuginosus脂酶基因：19671(SEQ ID NO：21)和991213J5(SEQ ID NO：22)。引物991213J5将SacII位点引入PCR片段中。利用BamHI和SacII切割PCR片段(约1100bp)并克隆入用相同酶切割的pEni1902中。通过DNA测序证实克隆。将质粒转化进大肠杆菌DH5α，脂酶的表达通过使用所述的滤膜法检测。
使用新开发的质粒有可能在没有任何修饰的曲霉属菌株中表达所需的酶。在大肠杆菌中获得的表达率相当低，但足以够筛选试验。
实施例2：制备热稳定的脂酶变体
应用几项技术在T.lanuginosus脂酶基因中产生多样性(diversity)：易错PCR，掺杂寡核苷酸辅助的定域随机诱变，和定点诱变。
显示更高温度稳定性的变体通过上面所述的初次筛选进行筛选，在LB培养基中培养并如上第二次筛选试验所述在试验板上再次划线。第二次筛选试验温度要高1-1.5度。对在这些条件下仍然具有活性的突变体的DNA进行测序并转化入曲霉以获得更高量的蛋白质，接着层析纯化。纯化的酶用于DSC分析以证明增加了稳定性。
接着，将在有利变体中发现的氨基酸取代组合，使用饱和诱变以确保所有的20种氨基酸在所需的位置引入。
实施例3：脂酶变体的热稳定性
实施例2中初次和第二次筛选中鉴定为热稳定性高的所有样品纯化至同质，并且它们的稳定性通过差示扫描热量测定(DSC)在pH 5.0和/或7.0检测以测定蛋白质的稳定性，通过熔解温度(TM)给定。包括T.lanuginosus亲代脂酶进行比较。
发现8种变体在pH 5.0的热稳定性升高，4种变体显示热稳定性升高在4℃以上。在pH 7.0检测两种变体发现具有改进的热稳定性。
实施例4：通过DSC确定脂酶变体的热稳定性
制备大量的T.lanuginosus脂酶变体并进行纯化，通过差示扫描热量测定(DSC)在pH 5.0检测热稳定性以测定蛋白质的稳定性，通过熔解温度(TM)给定。包括T.lanuginosus亲代脂酶用于比较。
发现下面的变体的热稳定性比亲代脂酶高：
| D111G+S216P |
| D27N |
| L227F |
| S224I+G225W+T226N+L227P+V228C |
| L227F+V228I |
| G225P |
| W221C+G246C |
发现下面的变体的热稳定性比亲代脂酶的熔解温度至少升高4℃。
| D27R+D111G+S216P |
| D27N+D111A |
| D27R+D111G+S216P+L227G+P256T |
| D27R+D111G+S216P+L227F+P256T |
| D27R+D111G+S216P+L227G |
| D27S+D111G+S216P |
| D27R+D111A+S216P+L227G+P256T |
| D27R+D111G+S216P+G225P+L227G+P256T |
| D27R+T37S+D111G+S216P+L227G+P256T |
| D27R+N39F+D111G+S216P+L227G+P256T |
| D27R+G38C+D111G+S216P+L227G+P256T |
| D27R+D111G+S216P+L227G+T244I+P256T |
| D27R+G91A+D111G+S216P+L227G+P256T |
| N25I+D27R+D111A+S216P+L227G+P256T |
| N25L+D27R+D111A+S216P+L227G+P256T |
| N26D+D27R+D111A+S216P+L227G+P256T |
| D27R+K46R+D111A+S216P+L227G+P256T |
| D27R+V60N+D111A+S216P+L227G+P256T |
| D27R+D111A+P136A+S216P+L227G+P256T |
| D27R+D111A+S216P+L227G+P256T+I265F |
| D27R+S58Y+D111A+S216P+L227G+P256T+ |
| N26D+D27R+E56Q+D111A+S216P+L227G+P256T |
| D27R+G91A+D96E+L97Q+D111A+S216P+L227G+P256T |
| D27R+G91A+D111A+S216P+L227G+P256T+ |
| D27R+G91T+N94S+D111A+S216P+L227G+P256T |
| D27R+G91S+D111A+S216P+L227G+P256T+ |
| D27R+G91N+D111A+S216P+L227G+P256T |
| D27R+D96E+D111A+S216P+L227G+P256T |
| D27R+I90L+G91A+N94K+D111A+S216P+L227G+P256T |
| D27R+G91S+F95V+D111A+S216P+L227G+P256T |
实施例5：平板鉴定热稳定性
制备大量的T.lanuginosus脂酶变体并如上“初次筛选实验”所述的检测热稳定性。包括T.la-nuginosus亲代脂酶用于比较。
发现下面的变体的热稳定性比亲代脂酶高：
| D27R+I90V+G91S+D111A+S216P+L227G+P256T |
| D27R+G91N+N94R+D111A+S216P+L227G+P256T |
| D27R+I90L+L93F+D96N+D111A+S216P+L227G+P256T |
| D27R+I90L+G91A+D96E+D111A+S216P+L227G+P256T |
| D27R+G91S+L93F+D111A+S216P+L227G+P256T |
| D27R+G91T+N94K+D111A+S216P+L227G+P256T |
| D27R+G91T+D111A+S216P+L227G+P256T |
| D27R+L93F+D111A+D137N+S216P+L227G+P256T |
| D27R+G91S+D96N+D111A+S216P+L227G+P256T |
| D27R+G91W+D111A+S216P+L227G+P256T |
| D27R+I90L+G91T+D111A+S216P+L227G+P256T |
| D27R+G91S+L93F+N94R+D96G+D111A+S216P+L227G+ |
| P256T |
| D27R+G91T+D96N+D111A+S216P+L227G+P256T |
| D27R+I90V+G91T+L93F+N94K+D111A+S216P+L227G+P256T |
| D27R+L93V+D111A+S216P+L227G+P256T |
| D27R+G91S+N94K+D111A+S216P+L227G+P256T |
| D27R+I90L+G91T+D111A+S216P+L227G+P256T |
| D27R+G91S+L93F+F95I+D96N+D111A+S216P+L227G+P256T |
| D27R+D111A+V187I+S216P+L227G+P256T |
| D27R+D111A+F211Y+S216P+L227G+P256T |
| D27R+R118M+D111A+A131V+S216P+L227G+P256T |
| D27R+P29S+R84C+D111A+H135Y+S216P+L227G+P256T |
| D27R+T32S+D111A+H135Y+S216P+L227G+P256T |
| D27R+G91R+D111A+I238V+S216P+L227G+P256T |
| D27R+F51I+I76V+N101D+D111A+N162R+S216P+L227G+P256T |
| D27R+F51L+D111A+S216P+L227G+P256T |
序列表
<110>诺维信公司(Novozymes A/S)
<120>脂解酶变体
<130>10133
<160>22
<170>PatentIn version 3.0
<210>1
<211>269
<212>PRT
<213>Thermomyces lanuginosus
<400>1
Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr
1 5 10 15
Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr
20 25 30
Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp
35 40 45
Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr
50 55 60
Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe
65 70 75 80
Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp
85 90 95
Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly
100 105 110
Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val
115 120 125
Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly
130 135 140
His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg
145 150 155 160
Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val
165 170 175
Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr
180 185 190
Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro
195 200 205
Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser
210 215 220
Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly
225 230 235 240
Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro
245 250 255
Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu
260 265
<210>2
<211>269
<212>PRT
<213>米赫根毛霉(Rhizomucor miehei)
<400>2
Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu
1 5 10 15
Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val
20 25 30
Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp
35 40 45
Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala
50 55 60
Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg
65 70 75 80
Gly Ser Ser Ser Ile Arg Asn Ala Ile Ala Asp Leu Thr Phe Val Pro
85 90 95
Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu
100 105 110
Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp
115 120 125
Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser
130 135 140
Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg
145 150 155 160
Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln
165 170 175
Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly
180 185 190
Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu
195 200 205
Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile
210 215 220
Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu
225 230 235 240
Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp
245 250 255
His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Ser
260 265
<210>3
<211>31
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>142779
<400>3
ttgaattgaa aatagattga tttaaaactt c 31
<210>4
<211>25
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>142780
<400>4
ttgcatgcgt aatcatggtc atagc 25
<210>5
<211>26
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>140288
<400>5
ttgaattcat gggtaataac tgatat 26
<210>6
<211>32
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>142778
<400>6
aaatcaatct attttcaatt caattcatca tt 32
<210>7
<211>11
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>gtactaaaacc
<400>7
gtactaaaac c 11
<210>8
<211>11
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>ccgttaaattt
<400>8
ccgttaaatt t 11
<210>9
<211>45
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>141223
<400>9
ggatgctgtt gactccggaa atttaacggt ttggtcttgc atccc 45
<210>10
<211>14
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>atgcaatttaaact
<400>10
atgcaattta aact 14
<210>11
<211>14
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>cggcaatttaacgg
<400>11
cggcaattta acgg 14
<210>12
<211>44
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>141222
<400>12
ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc 44
<210>13
<211>26
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>270999J8
<400>13
tctgtgaggc ctatggatct cagaac 26
<210>14
<211>27
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>270999J9
<400>14
gatgctgcat gcacaactgc acctcag 27
<210>15
<211>59
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>051199J1
<400>15
cctctagatc tcgagctcgg tcaccggtgg cctccgcggc cgctggatcc ccagttgtg 59
<210>16
<211>33
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>1298TAKA
<400>16
gcaagcgcgc gcaatacatg gtgttttgat cat 33
<210>17
<211>30
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>177996
<400>17
gaatgacttg gttgacgcgt caccagtcac 30
<210>18
<211>25
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>().()
<223>135640
<400>18
cttattagta ggttggtact tcgag 25
<210>19
<211>37
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>135638
<400>19
gtccccagag tagtgtcact atgtcgaggc agttaag 37
<210>20
<211>64
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>080399J19
<400>20
gtatgtccct tgacaatgcg atgtatcaca tgatataatt actagcaagg gaagccgtgc 60
ttgg 64
<210>21
<211>24
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>19671
<400>21
ctcccttctc tgaacaataa accc 24
<210>22
<211>66
<212>DNA
<213>人工的/未知
<220>
<221>misc_feature
<222>()..()
<223>991213J5
<400>22
cctctagatc tcgagctcgg tcaccggtgg cctccgcggc cgctgcgcca ggtgtcagtc 60
accctc 66
Claims (27)
1.一种亲代真菌脂解酶的变体，其中该变体
a)所具有的氨基酸序列与SEQ ID NO：1具有至少80％的同源性，和与亲代真菌脂解酶相比，所具有的氨基酸序列包括相应于SEQ ID NO：1的P29S，T32S，F51I/L，R84C，I90L，G91N/W，L93F，F951，N101D，R118M，A131V，H135Y，N162R，V187I，S224I/Y，T226N，L227F/P/G/V，V228C或I238V的一个或更多个取代，和
b)比亲代脂解酶的热稳定性高。
2.权利要求1的变体，其比亲代脂解酶的热稳定性至少高4℃。
3.权利要求1或2的变体，其中亲代脂解酶与SEQ ID NO：1具有至少50％的同源性。
4.权利要求1或2的变体，其中亲代脂解酶具有SEQ ID NO：1所示的氨基酸序列。
5.权利要求1或2的变体，具有1，2，3，4，5，6，7或8个所述的取代。
6.权利要求1或2的变体，进一步包括一个或多个不是权利要求1中所列那些的氨基酸残基取代。
7.权利要求6的变体，该取代是1-5个。
8.权利要求1或2的变体，包括相应于SEQ ID NO：1中的下述取代：
a)D27N
b)D111G+S216P
c)L227F
d)L227F+V228I
e)G225P
f)S224I+G225W+T226N+L227P+V228C
g)S224Y+G225W+T226N+L227P+V228C
h)D27R+D111G+S216P
i)D27S+D111G+S216P
j)D27N+D111A
k)D27R+D111G+S216P+L227P+P256T
l)D27R+D111G+S216P+L227G+P256T
m)D27R+D111G+S216P+L227F+P256T
n)D27R+D111G+S216P+L227V+P256T
o)D27R+D111G+S216P+L227G
p)D27R+D111G+S216P+L227X
q)D27P+D111G+S216P+L227X
r)S224I+G225W+T226N+L227P+V228C
s)W221C+G246C
t)D27R+D111G+S216P
u)D27N+D111A
v)D27R+D111G+S216P+L227G+P256T
w)D27R+D111G+S216P+L227F+P256T
x)D27R+D111G+S216P+L227G
y)D27S+D111G+S216P
z)D27R+D111A+S216P+L227G+P256T
aa)D27R+D111G+S216P+G225P+L227G+P256T
bb)D27R+T37S+D111G+S216P+L227G+P256T
cc)D27R+N39F+D111G+S216P+L227G+P256T
dd)D27R+G38C+D111G+S216P+L227G+P256T
ee)D27R+D111G+S216P+L227G+T244I+P256T
ff)D27R+G91A+D111G+S216P+L227G+P256T
gg)N25I+D27R+D111A+S216P+L227G+P256T
hh)N25L+D27R+D111A+S216P+L227G+P256T
ii)N26D+D27R+D111A+S216P+L227G+P256T
jj)D27R+K46R+D111A+S216P+L227G+P256T
kk)D27R+V60N+D111A+S216P+L227G+P256T
ll)D27R+D111A+P136A+S216P+L227G+P256T
mm)D27R+D111A+S216P+L227G+P256T+I265F
nn)D27R+S58Y+D111A+S216P+L227G+P256T+
oo)N26D+D27R+E56Q+D111A+S216P+L227G+P256T
pp)D27R+G91A+D96E+L97Q+D111A+S216P+L227G+P256T
qq)D27R+G91A+D111A+S216P+L227G+P256T+
rr)D27R+G91T+N94S+D111A+S216P+L227G+P256T
ss)D27R+G91S+D111A+S216P+L227G+P256T+
tt)D27R+G91N+D111A+S216P+L227G+P256T
uu)D27R+D96E+D111A+S216P+L227G+P256T
vv)D27R+I90L+G91A+N94K+D111A+S216P+L227G+P256T
ww)D27R+G91S+F95V+D111A+S216P+L227G+P256T。
9.权利要求1或2的变体，具有的变性温度比亲代脂解酶至少高5℃。
10.权利要求9的变体，具有的变性温度在PH 5-7测定。
11.一种编码任何前述权利要求之一变体的DNA。
12.一种包括权利要求11所述的DNA的载体。
13.一种携带有权利要求11的DNA或权利要求12的载体的转化宿主细胞。
14.一种制备权利要求1-10中任一项的变体的方法，包括
a)培养权利要求13的细胞以表达该变体，和
b)回收该变体。
15.权利要求14所述的方法，其中在a)中分泌该变体。
16.一种制备脂解酶变体的方法，包括：
a)筛选亲代真菌脂解酶，
b)在亲代脂解酶中进行至少一个下述取代，相应于SEQ ID NO：1中P29S，T32S，F51I/L，R84C，I90L，G91N/W，L93F，F95I，N101D，R118M，A131V，H135Y，N162R，V187I，S224I/Y，T226N，L227F/P/G/V，V228C或I238V
c)可选地，取代一个或多个不同于b)中的氨基酸，其所具有的氨基酸序列与SEQ ID NO：1具有至少80％的同源性，
d)制备由步骤a)-c)而得的变体，
e)检测该变体的热稳定性，
f)筛选具有增高热稳定性的变体，和
g)制备所筛选的变体。
17.权利要求16所述的方法，其中亲代脂解酶与SEQ ID NO：1具有至少50％的同源性。
18.权利要求16所述的方法，其中亲代脂解酶具有SEQ ID NO：1所示的序列。
19.一种水解羧酸酯的方法，包括将该酯与权利要求1-10中任一项的脂解酶的变体在有水的情况下温育。
20.一种在制备机械纸浆或使用机械纸浆造纸的方法中控制树脂问题的方法，包括将权利要求1-10中任一项的脂解酶的变体添加到纸浆中并温育。
21.权利要求20所述的方法，其中所述温育是在温度60-95℃进行。
22.权利要求21所述的方法，其中所述温度是在75-90℃进行。
23.权利要求20所述的方法，其中温育在PH 4.5-11进行。
24.权利要求23所述的方法，其中温育在5-6.5进行。
25.一种制备生面团或由生面团制备焙烤产品的方法，包括将权利要求1-10中任一项的脂解酶变体添加到生面团中。
26.一种水解，合成或酯交换酯的方法，包括在权利要求1-10中任一项的脂解酶的变体存在下酯与水的反应，酸与醇的反应或酯与酸，醇或第二种酯的酯交换。
27.一种从织物中用酶去除疏水性酯的方法，包括用有效量的权利要求1-10中任一项的脂解酶的变体处理织物以从织物中去除疏水性酯。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DKPA200100032 | 2001-01-10 | ||
| DKPA200100032 | 2001-01-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1484693A CN1484693A (zh) | 2004-03-24 |
| CN100529069C true CN100529069C (zh) | 2009-08-19 |
Family
ID=32087885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB028035852A Expired - Lifetime CN100529069C (zh) | 2001-01-10 | 2002-01-10 | 脂解酶变体 |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US20040152180A1 (zh) |
| CN (1) | CN100529069C (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865896A (zh) * | 2014-03-11 | 2014-06-18 | 上海康地恩生物科技有限公司 | 一种碱性脂肪酶突变体 |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101541956B (zh) * | 2006-11-28 | 2012-04-18 | 诺维信公司 | 脂解酶变体 |
| MX2010009072A (es) * | 2008-02-29 | 2010-09-24 | Novozymes As | Polipeptidos que tienen actividad de lipasa y polinucleotidos que codifican para los mismos. |
| US9909109B2 (en) * | 2012-04-02 | 2018-03-06 | Novozymes A/S | Lipase variants and polynucleotides encoding same |
| AU2013328953A1 (en) * | 2012-10-12 | 2015-03-26 | Danisco Us Inc. | Compositions and methods comprising a lipolytic enzyme variant |
| EP3696264B1 (en) * | 2013-07-19 | 2023-06-28 | Danisco US Inc. | Compositions and methods comprising a lipolytic enzyme variant |
| EP3760713A3 (en) * | 2014-05-27 | 2021-03-31 | Novozymes A/S | Lipase variants and polynucleotides encoding same |
| WO2016091870A1 (en) * | 2014-12-09 | 2016-06-16 | Novozymes A/S | Lipase variants and polynucleotides encoding same |
| EP3929285A3 (en) | 2015-07-01 | 2022-05-25 | Novozymes A/S | Methods of reducing odor |
| CN117448299A (zh) * | 2017-09-27 | 2024-01-26 | 诺维信公司 | 脂肪酶变体和包含此类脂肪酶变体的微囊组合物 |
| CN111378585B (zh) * | 2018-12-28 | 2023-06-16 | 丰益(上海)生物技术研发中心有限公司 | 用于表达外源基因的毕赤酵母突变株 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5869438A (en) * | 1990-09-13 | 1999-02-09 | Novo Nordisk A/S | Lipase variants |
| ATE326572T1 (de) * | 1991-12-20 | 2006-06-15 | Novozymes As | Entfernung von hydrophoben estern aus textilien |
-
2002
- 2002-01-10 US US10/250,522 patent/US20040152180A1/en not_active Abandoned
- 2002-01-10 CN CNB028035852A patent/CN100529069C/zh not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865896A (zh) * | 2014-03-11 | 2014-06-18 | 上海康地恩生物科技有限公司 | 一种碱性脂肪酶突变体 |
Also Published As
| Publication number | Publication date |
|---|---|
| US20040152180A1 (en) | 2004-08-05 |
| CN1484693A (zh) | 2004-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1352057B1 (en) | Lipolytic enzyme variant | |
| US7226770B2 (en) | Lipolytic enzyme variant | |
| AU2002219020A1 (en) | Thermostable lipolytic enzyme variant | |
| CN101374947B (zh) | 具有脂肪酶活性的多肽和编码该多肽的多核苷酸 | |
| CN101990573B (zh) | 具有改进的稳定性的脂肪分解酶变体及编码该变体的多核苷酸 | |
| CN100529069C (zh) | 脂解酶变体 | |
| CN105296445A (zh) | 脂肪酶变体 | |
| CA2467415A1 (en) | Lipolytic enzyme variants and method for their production | |
| AU2007217732B2 (en) | Novel Lipases and uses thereof | |
| AU2007304533B9 (en) | Clonation, expression and use of acid phospholipases | |
| WO2008074884A2 (en) | Novel xylanase enzymes xyl001 and xyl002 and uses thereof | |
| MX2008008925A (es) | Polipeptidos que tienen actividad de lipasa y polinucleotidos que codifican para los mismos |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20090819 |