AU7642398A - Phenolic acid esterase and use thereof - Google Patents
Phenolic acid esterase and use thereof Download PDFInfo
- Publication number
- AU7642398A AU7642398A AU76423/98A AU7642398A AU7642398A AU 7642398 A AU7642398 A AU 7642398A AU 76423/98 A AU76423/98 A AU 76423/98A AU 7642398 A AU7642398 A AU 7642398A AU 7642398 A AU7642398 A AU 7642398A
- Authority
- AU
- Australia
- Prior art keywords
- enzyme
- gly
- ggt
- asn
- ala
- 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.)
- Abandoned
Links
- 150000007965 phenolic acids Chemical class 0.000 title claims description 31
- 108010013043 Acetylesterase Proteins 0.000 title claims description 28
- 102000004190 Enzymes Human genes 0.000 claims description 115
- 108090000790 Enzymes Proteins 0.000 claims description 115
- 229940088598 enzyme Drugs 0.000 claims description 114
- 230000000694 effects Effects 0.000 claims description 31
- 238000002360 preparation method Methods 0.000 claims description 26
- ZZXREVQVZCYAIE-IGBVEJJSSA-N [(2s,3r,4r)-5-[(2s,3r,4s,5r)-3,5-dihydroxy-2-[(2r,3r,4r)-1,3,4-trihydroxy-5-oxopentan-2-yl]oxyoxan-4-yl]oxy-3,4-dihydroxyoxolan-2-yl]methyl (e)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)OC[C@H]2[C@@H]([C@@H](O)C(O[C@@H]3[C@H]([C@H](O[C@H](CO)[C@H](O)[C@@H](O)C=O)OC[C@H]3O)O)O2)O)=C1 ZZXREVQVZCYAIE-IGBVEJJSSA-N 0.000 claims description 19
- 239000000758 substrate Substances 0.000 claims description 18
- 108020004414 DNA Proteins 0.000 claims description 17
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 102000053602 DNA Human genes 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 9
- 229920001282 polysaccharide Polymers 0.000 claims description 8
- 239000005017 polysaccharide Substances 0.000 claims description 8
- 150000004804 polysaccharides Chemical class 0.000 claims description 8
- 230000000593 degrading effect Effects 0.000 claims description 7
- 108010041969 feruloyl esterase Proteins 0.000 claims description 7
- 241000422444 Piromyces equi Species 0.000 claims description 6
- 239000001913 cellulose Substances 0.000 claims description 6
- 229920002678 cellulose Polymers 0.000 claims description 6
- -1 a-galactosidase Proteins 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 5
- 235000019800 disodium phosphate Nutrition 0.000 claims description 5
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 4
- 239000003674 animal food additive Substances 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 235000009048 phenolic acids Nutrition 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 235000000346 sugar Nutrition 0.000 claims description 3
- 108010011619 6-Phytase Proteins 0.000 claims description 2
- 239000004382 Amylase Substances 0.000 claims description 2
- 102000013142 Amylases Human genes 0.000 claims description 2
- 108010065511 Amylases Proteins 0.000 claims description 2
- 102100032487 Beta-mannosidase Human genes 0.000 claims description 2
- 108090000604 Hydrolases Proteins 0.000 claims description 2
- 102000004157 Hydrolases Human genes 0.000 claims description 2
- 108090001060 Lipase Proteins 0.000 claims description 2
- 102000004882 Lipase Human genes 0.000 claims description 2
- 239000004367 Lipase Substances 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- 102100036617 Monoacylglycerol lipase ABHD2 Human genes 0.000 claims description 2
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 241000193632 Piromyces sp. Species 0.000 claims description 2
- 108010059820 Polygalacturonase Proteins 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 2
- 108010093941 acetylxylan esterase Proteins 0.000 claims description 2
- 235000019418 amylase Nutrition 0.000 claims description 2
- 108010055059 beta-Mannosidase Proteins 0.000 claims description 2
- 108010080434 cephalosporin-C deacetylase Proteins 0.000 claims description 2
- 244000038559 crop plants Species 0.000 claims description 2
- 235000019621 digestibility Nutrition 0.000 claims description 2
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 2
- 125000003147 glycosyl group Chemical group 0.000 claims description 2
- 235000019421 lipase Nutrition 0.000 claims description 2
- 244000144972 livestock Species 0.000 claims description 2
- 229940085127 phytase Drugs 0.000 claims description 2
- 150000008163 sugars Chemical class 0.000 claims description 2
- 108010060309 Glucuronidase Proteins 0.000 claims 1
- 102000053187 Glucuronidase Human genes 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 46
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 32
- 108010078144 glutaminyl-glycine Proteins 0.000 description 27
- 108010090461 DFG peptide Proteins 0.000 description 20
- WJZLEENECIOOSA-WDSKDSINSA-N Gly-Asn-Gln Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)O WJZLEENECIOOSA-WDSKDSINSA-N 0.000 description 17
- 241000196324 Embryophyta Species 0.000 description 15
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 15
- 108010038745 tryptophylglycine Proteins 0.000 description 14
- BYXHQQCXAJARLQ-ZLUOBGJFSA-N Ala-Ala-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O BYXHQQCXAJARLQ-ZLUOBGJFSA-N 0.000 description 13
- KVNOBVKRBOYSIV-SZMVWBNQSA-N Met-Pro-Trp Chemical compound CSCC[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N KVNOBVKRBOYSIV-SZMVWBNQSA-N 0.000 description 12
- QJHOOKBAHRJPPX-QWRGUYRKSA-N Asp-Phe-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 QJHOOKBAHRJPPX-QWRGUYRKSA-N 0.000 description 10
- RPLLQZBOVIVGMX-QWRGUYRKSA-N Gly-Asp-Phe Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RPLLQZBOVIVGMX-QWRGUYRKSA-N 0.000 description 10
- QPCVIQJVRGXUSA-LURJTMIESA-N Gly-Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QPCVIQJVRGXUSA-LURJTMIESA-N 0.000 description 10
- 210000002421 cell wall Anatomy 0.000 description 10
- LPAJOCKCPRZEAG-MNXVOIDGSA-N Lys-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCCCN LPAJOCKCPRZEAG-MNXVOIDGSA-N 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 8
- VJTWLBMESLDOMK-WDSKDSINSA-N Asn-Gln-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O VJTWLBMESLDOMK-WDSKDSINSA-N 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 229920001221 xylan Polymers 0.000 description 7
- 150000004823 xylans Chemical class 0.000 description 7
- NITWSHWHQAQBAW-QPJJXVBHSA-N (E)-4-coumaric acid methyl ester Chemical compound COC(=O)\C=C\C1=CC=C(O)C=C1 NITWSHWHQAQBAW-QPJJXVBHSA-N 0.000 description 6
- 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 6
- RGNMNWULPAYDAH-JSGCOSHPSA-N Gln-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N RGNMNWULPAYDAH-JSGCOSHPSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 108010051242 phenylalanylserine Proteins 0.000 description 6
- AUJXJFHANFIVKH-GQCTYLIASA-N trans-methylferulate Chemical compound COC(=O)\C=C\C1=CC=C(O)C(OC)=C1 AUJXJFHANFIVKH-GQCTYLIASA-N 0.000 description 6
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 description 5
- 229940114124 ferulic acid Drugs 0.000 description 5
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 description 5
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 description 5
- 235000001785 ferulic acid Nutrition 0.000 description 5
- 229920005610 lignin Polymers 0.000 description 5
- 108010005942 methionylglycine Proteins 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 description 5
- MIPWEZAIMPYQST-FXQIFTODSA-N Ala-Cys-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O MIPWEZAIMPYQST-FXQIFTODSA-N 0.000 description 4
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 4
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 4
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 4
- WUQXMTITJLFXAU-JIOCBJNQSA-N Asn-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N)O WUQXMTITJLFXAU-JIOCBJNQSA-N 0.000 description 4
- DTNUIAJCPRMNBT-WHFBIAKZSA-N Asp-Gly-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O DTNUIAJCPRMNBT-WHFBIAKZSA-N 0.000 description 4
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 4
- JNNVNVRBYUJYGS-CIUDSAMLSA-N Asp-Leu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O JNNVNVRBYUJYGS-CIUDSAMLSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- XSBGUANSZDGULP-IUCAKERBSA-N Gln-Gly-Lys Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCCCN)C(O)=O XSBGUANSZDGULP-IUCAKERBSA-N 0.000 description 4
- SOEXCCGNHQBFPV-DLOVCJGASA-N Gln-Val-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SOEXCCGNHQBFPV-DLOVCJGASA-N 0.000 description 4
- CIMULJZTTOBOPN-WHFBIAKZSA-N Gly-Asn-Asn Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CIMULJZTTOBOPN-WHFBIAKZSA-N 0.000 description 4
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- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 4
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- QHDXUYOYTPWCSK-RCOVLWMOSA-N Val-Asp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N QHDXUYOYTPWCSK-RCOVLWMOSA-N 0.000 description 4
- CPGJELLYDQEDRK-NAKRPEOUSA-N Val-Ile-Ala Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](C)C(O)=O CPGJELLYDQEDRK-NAKRPEOUSA-N 0.000 description 4
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- 235000013311 vegetables Nutrition 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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Description
WO 98/46768 PCT/EP98/02080 Phenolic Acid Esterase and Use Thereof The present invention relates to an enzyme with phenolic acid esterase activity, DNA molecule encoding said enzyme as well as a method for the production and use of said enzyme. Background Plant cell walls are divided into two sections, the primary and secondary cell wall. The primary cell wall is comprised of three major classes of polysaccharides: cellulose, hemicellulose and pectin. The secondary cell wall also contains polysaccharides as well as lignin. Hemicellulose, a general class of highly branched polysaccharides found in the plant cell wall, is bound to itself as well as to cellulose and lignin, and these bonds contribute to the stabili-ty and support of the plant structure. Hemicelluloses based on a xylose backbone are designated as xylans. Xylan, which has been shown to exist in a wide variety of different plants including fruits, vegetables legumes, cereals, grasses, softwoods and hardwoods, is a linear 0-(1-4)-D-xylopyranose polymer which can be substituted with sugar residues, including a-L-arabinose, and a-D glucuronic acid and/or the 4-0-methyl ether derivative of a-D glucuronic acid. Many xylans are also esterified with phenolic acid residues, including coumaric acid and ferulic acid. These phenolic acid residues are present in an ester linkage to a-L-arabinofuranosyl xylan and can serve to protect xylan from xylan-degrading enzymes, so-called xylanases, as well they confer structural stability to the plant cell wall by forming covalent bonds with the lignin present therein. In addition, ferulic acid has been shown to exist as a diferulic acid bridge between different xylan chains, imparting further structural support for plant cells (Linden, J.C. et al., American Chemical Society Symposium Series, vol. 566 (1994), 452-467).
WO 98/46768 PCT/EP98/02080 2 A number of microorganisms are known which are capable of hydrolysing phenolic acid esters and digesting plant cell walls through the enzymatic breakdown of plant cell wall polysaccharides. Some of these microorganisms possess enzyme(s) with phenolic acid esterase activity, i.e. coumaric acid esterase activity or ferulic acid esterase activity or a combination of these two activities. For example, Borneman, W.S. et al (Applied and Environmental Microbiology, vol. 58 (1992), 3762-3766) describe two ferulic acid esterases (FAE), designated FAE-I and FAE-II respectively, isolated from the anaerobic fungus Neocallimastix strain MC-2. FAE-II was reported to be specific for the substrate (O-{5-0-[(E)-feruloyl]-a-L arabinofuranosyl}-(1-3)-O--D-xylopyranosyl-(1-4)-D- xylopyranose (FAXX), whereas FAE-I was reported to have both a FAXX degrading activity as well as a (0-{5-0- [ (E) -p coumaroyll -a-L-arabinofuranosyl}- (1-3) -O-0-D-xylopyranosyl- (1 4) -D-xylopyranose (PAXX) degrading activity, the maximum ratio of metabolism of FAXX:PAXX being 3:1. The pH optima of these two enzymes were shown to be 6.2 and 7.0 respectively when using FAXX as a substrate. GB 2 301 103 discloses an FAE obtained from Aspergillus niger as well as the gene encoding said enzyme. Said enzyme has a pH optimum of about 5 and a temperature optimum of from about 50 to 60 0 C when methyl ferulate is used as a substrate. Other purified enzymes with ferulic acid esterase activity are known (for example, see McCrae, S.I. et al., Enzyme Microb. Technol., vol. 16 (1994), 826-834; Faulds, C.B. and Williamson, G., Microbiology, vol. 140 (1994), 779-787; Castanares, A. et al., Enzyme Microb. Technol., vol. 14 (1992), 875; and Kroon, P.A. et al., Biotechnol. Appl. Biochem., vol 23 (1996), 255-262) which have pH optima ranging from about 5.0 to 6.0 and temperature optima from 30 to 60 0
C.
WO 98/46768 PCT/EP98/02080 3 Enzymes with phenolic acid esterase activity can be used in a number of industrial, agricultural and health applications which can be carried out at pH values about or above 6.5 and/or at temperatures above 45 0 C. Summary of the invention It is an object of the present invention to provide enzyme with good phenolic acid esterase activity. In addition, it is an object of the present invention to provide a source of an enzyme with phenolic acid esterase activity which is available in relatively large amounts. Furthermore, it is an object to provide a method for the production of an enzyme with phenolic acid esterase activity. A further object is to provide uses of an enzyme phenolic acid esterase activity for the preparation of food and feed, for the processing of paper and pulp as well as for the bioconversion of ligno-cellulose wastes, for example. Other objects of the present invention will become apparent from the following detailed description. Subject matter of the present invention is an enzyme with phenolic acid esterase activity, characterized in that said enzyme has a pH optimum greater than pH 6.5, preferably about 7.0, and a temperature optimum greater than 45 0 C, preferably greater than 50 0 C, and more preferably about 55 0 C, when measured in a citric acid/disodium hydrogen orthophosphate buffer containing 33 pM FAXX as a substrate. Preferably, said enzyme has ferulic acid esterase activity and coumaric acid esterase activity.
WO 98/46768 PCT/EP98/02080 4 Subject matter of the present invention is also an enzyme with phenolic acid esterase activity obtainable from Piromyces Sp., for example Piromyces equi, and more preferably from the Piromyces equi strain deposited under the Budapest Treaty at the International Mycological Institute (IMI), Bakeham Lane, Egham, Surrey, UK under the Accession Number 375061. Preferably, the enzyme of the present invention comprises the amino acid sequence given in SEQ ID NO:1 or a functional derivative thereof. A functional derivative of the enzyme of the present invention is defined as an enzyme having one or more N-terminal, C-terminal or internal substitution(s), insertion(s) and/or deletion(s) in the amino acid sequence given in SEQ ID NO:1 which maintains a pH optimum greater than 6.5, preferably about 7.0, and a temperature optimum greater than 45 0 C, preferably greater than 50"C, and more preferably about 55 0 C, when measured in a citric acid/disodium hydrogen orthophosphate buffer containing 33 [M FAXX as a substrate. More preferably, the enzyme of the present invention comprises the amino acid sequence given in SEQ ID NO:3 or a functional derivative thereof. In addition, the enzyme of the present invention is preferably encoded by the DNA sequence given in SEQ ID NO:1 or a functional derivative thereof. More preferably, the enzyme of the present invention is encoded by the DNA sequence given in SEQ ID NO:3 or a functional derivative thereof. The present invention relates to a phenolic acid esterase with one or more of the above properties. Further subject matter of the present invention is a DNA molecule encoding an enzyme with phenolic acid esterase activity, characterized in that said DNA molecule comprises a DNA sequence as given in SEQ ID NO:1 or a functional derivative or homologue thereof. A functional derivative of the DNA sequence given in SEQ ID NO:1 is defined as a DNA WO 98/46768 PCT/EP98/02080 5 sequence having one or more 5'-, 3'- or internal substitution (s) , insertion (s) and/or deletion (s) in the DNA sequence given in SEQ ID NO:1 which maintains its capability to encode an enzyme with phenolic acid esterase activity which has a pH optimum greater than 6.5, preferably about 7.0, and a temperature optimum greater than 45 0 C, preferably greater than 50'C, and more preferably about 55 0 C, when measured in a citric acid/disodium hydrogen orthophosphate buffer comprising 33 pM FAXX as a substrate. A functional homologue of the DNA sequence of the present invention is defined as a DNA sequence with preferably 75% homology, more preferably 85% homology and most preferably 95% homology to the DNA sequence given in SEQ ID NO:1 or SEQ ' ID NO:3. More preferably, a DNA molecule encoding an enzyme according to the present invention comprises a DNA sequence as given in SEQ ID NO:3 or a functional derivative or homologue thereof. In a preferred embodiment, DNA molecules of the present invention comprise vector sequence capable of replicating said DNA molecules and/or expressing said enzyme in a procaryotic or eucaryotic host. Further subject matter of the present invention is a transformed procaryotic cell or eucaryotic cell comprising one or more DNA molecules of the present invention. Preferably said cells are selected from the group comprising E. coli, Bacillus sp., such as Bacillus subtilis, Lactobacillus sp., and Lactococcus sp., Aspergillus, Trichoderma, Penicillium, Mucor, Kluyveromyces and Saccharomyces, such as Saccharomyces cerevisiae. The enzyme of the present invention may be expressed in transgenic plants such as maize, soybean and canola/rapeseed. or in root storage organs of plants, such as potato, carrot and sugar beet.
WO 98/46768 PCT/EP98/02080 6 The introduction of an esterase of the present invention expressed and/or secreted at the appropriate stage, for example, at harvest, has the advantage that the risk of weakening the transgenic plant or storage root organ structure during growth can be reduced. The methodology for the production of transformed procaryotic and eucaryotic cells is known in the art. Transgenic fungus, such as Aspergillus, tranformed yeast, such as Saccharomyces, and transgenic plants are also known inthe art and can be produced by the methods taught and discussed in GB 2 301 103, EP 479 359 and EP 449 375. Subject matter of the present invention is also a method for the production of an enzyme or enzyme preparation having phenolic acid esterase activity, characterized in that said enzyme is isolated from a naturally occurring organism or transformed cell or organism capable of expressing the enzyme according to the invention. Enzyme preparations including, for example, partially purified preparations obtainable as a cell or organism extract are also subject matter of the present invention. The enzyme preparation of the present invention can comprise one or more further polysaccharide modifying and/or degrading enzymes. Said polysaccharide modifying and/or degrading enzyme(s) is (are) preferably selected from the group comprising xylanase, arabinanase, a-L-arabinofuranosidase, endoglucanase, c-D-glucuronidase, pectinase, acetyl esterase, mannanase, acetyl xylan esterase and other glycosyl hydrolases. In addition, the enzyme preparation of the present invention can also include one or more further enzymes selected from the group comprising amylase, protease, a-galactosidase, phytase and lipase.
WO 98/46768 PCT/EP98/02080 7 Use of the enzyme and/or enzyme preparation according to the invention include the use in a process for releasing phenolic acids from a substrate comprising phenolic acid moieties. Said enzyme and/or enzyme preparation according to the invention can equally find use in the production of animal feed by improving the digestibility of plant material, especially forage in which the plant cell walls have a high phenolic acid content. Furthermore, the enzyme and/or the enzyme preparation according to the invention can be used in or with crop plants including but not limited to maize, wheat, grasses and alfalfa, to improve the digestability for livestock by pre-modifying the cell wall content. Said enzyme and/or enzyme preparation according to the invention can also find used in the preparation of food for human consumption. Further subject matter of the present invention is also a feed additive comprising an enzyme or enzyme preparation having phenolic acid esterase activity according to the invention and a feed comprising said feed additive. The enzyme and/or enzyme preparation according to the invention can also find use in the paper and pulp industry, for example, in helping remove lignin from cellulose pulps. Additionally, used in combination with xylan degrading enzymes, the enzyme and/or enzyme preparation according to the invention can contribute to a reduction in the amount of chlorine required for bleaching by increasing the solubility and extractability of lignin from pulp. Furthermore, when combined with xylanases and/or cellulases, the enzyme and/or enzyme preparation according to the invention can be used for the bioconversion of plant material or ligno-cellulose wastes to sugars, for example, for chemical or fuel production, and/or in the production of phenolic acids.
WO 98/46768 PCT/EP98/02080 8 Brief description of the drawings Figure 1: pH profile of the phenolic acid esterase of the present invention measured using FAXX as a substrate. Figure 2: Temperature profile of the phenolic acid esterase of the present invention measured using FAXX as a substrate. Detailed description of the invention The following Examples are intended to more closely illustrate the present invention without limiting the subject matter of the invention to said Examples. Example 1 Piromyces equi isolated from horse cecum (Orpin, C.G., J. Gen. Microbiol., vol. 123 (1981), 287-296) and as described by E.A. Munn in Anaerobic Fungi, Biology, Ecology and Function, D.O. Mountfort and C.G. Orpin Eds., Marcel Dekker, Inc., New York, 1994, 47-105, and deposited under the Budapest Treaty at the International Mycological Institute (IMI) under the Accession Number 375061 was cultured under anaerobic conditions at a temperature of 39 0 C in a rumen fluid-containing medium (Kemp,P., Lander, D.J. and Orpin, C.G., J. Gen. Microbiol., vol. 130 (1984), 27-37) with 0.10% soluble xylan and 0.5% Sigmacell (Sigma Chemical Co., Poole, Dorset, England) as carbon sources. Total RNA was extracted from fungus grown under the above conditions, poly(A)+ RNA was selected by oligo(dT) chromatography, and double-stranded cDNA was synthesized from the selected RNA, cloned into XZAPII using a ZAP-cDNA synthesis kit and packaged in vitro according to the instructions of the manufacturer (Stratagene, La Jolla, California, USA) (Xue, G-P. et al., J Gen. Microbiol., vol. 138 (1992), 1413-1420 and Ali, B.R.S. et al., FEMS Microbiol.
WO 98/46768 PCT/EP98/02080 9 Lett., vol. 125 (1995), 15-22). Recombinant phage were grown by plating on lawns of E. coli XL1-Blue in soft agar overlays and screened using an antibody raised against a fungal cellulase/hemicellulase complex purified according to Ali,B.R.S. et al., FEMS Microbiol. Lett., vol. 125 (1995), 15 22) . Antibody screening of phage plaques with rabbit anti complex antibody as the primary antibody was carried out essentially as described in the instruction manual provided with the picoBlueTM immunoscreening kit (Stratagene) , with the following modifications: isopropyl-@-D-thiogalactopyranoside (IPTG: 0.33 mM) was added directly to the soft agar overlays containing recombinant XZAPII and host bacteria (E. coli XL1 Blue); plaques were lifted onto Hybond-C filters (Amersham); blocking solution contained dried milk powder (4% w/v) in place of BSA; anti-rabbit IgG (whole molecule) conjugated to horseradish peroxidase (Sigma Chemical Co.) was used as secondary antibody; colour development solution comprised 3,3'-diaminobenzidine (0.5 mg/ml) in 50 mM Tris-HC1 buffer, pH 7.4, containing hydrogen peroxide (0.5 pl/ml. Esterase production was verified by showing that a clone selected by antibody screening synthesized an enzyme which hydrolysed [4 methylumbelliferoyl (p-trimethylammonium cinnamate chloride)] according to Dalrymple, B.P. et al., FEMS Microbiol. Lett., vol 143 (1996), 115-120. General molecular biological techniques including DNA isolation, restriction endonuclease digestion, ligation, transformation as well as DNA sequencing of the esterase gene were performed in accordance with Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed. (1989), Cold Spring Harbor, New York. Nucleotide sequencing of the the gene encoding the enzyme having phenolic acid esterase activity of the present invention was performed and the results are given in SEQ ID NO:3. The open reading frame comprises 1608 nucleotides, WO 98/46768 PCT/EP98/02080 10 encoding a protein of 536 amino acids with a predicted molecular weight of 55,540 daltons. Example 2 Measurement of pH optimum A truncated enzyme encoded by SEQ ID NO. 1 was generated in a PCR reaction (20 cycles of 30 seconds at 94 0 C, 45 seconds at 50 0 C, and 1 minute at 72 0 C) in a buffer comprising 50 mM Tris HCl, pH 9.0, 50 mM NaCl, 10 mM MgCl 2 , 200 pM dNTPs, 50 picomoles of the primers 5' -end: 5 ' -CGCGGATCCAACAGCGGTCCAACTGTTG-3' 3 - end: 5' -GCGAATTCTTATCTTATGGGAGAGAG-3', and 250 ng template DNA and expressed in E.coli BL21 (DE3) (Novagen, Inc., Wisconsin, USA) using the vector pET32a (Novagen, Inc.). The enzyme was purified from freshly prepared cell-free extracts by binding to Talon resin (Clontech Laboratories Inc., California, USA) and cleaved from the metal affinity resin using restriction grade Thrombin (Sigma) in accordance to the guidelines provided by Novagen, Inc., USA, for use with pET vectors. The enzyme was further purified as follows: a 1 ml MonoQ column (Pharmacia) was equilibrated with 10 mM Tris, pH 8.0, and fresh enzyme was applied. The enzyme was eluted at 1.0 ml/min with a sodium chloride gradient (0 to 0.5 M NaCl in 10 mM Tris, pH 8.0) . Fractions of 1.0 ml were collected. The enzyme was assayed in McIlvaine's buffer (citric acid/ disodium hydrogen orthophosphate, see Data for Biochemical Research, 3rd Edition, Dawson, Elliot, Elliot, Jones, Oxford Science Publications, Oxford University Press, 1987) for pH values ranging from 3 to 7 or a buffer comprising potassium chloride/ boric acid for pH values ranging from 8 to 9. The assay was carried out at 37 0 C with a final FAXX concentration of 33 pM. Ferulic acid release from FAXX was monitored WO 98/46768 PCT/EP98/02080 11 continuously for 3 min at 335 nm according to (Faulds, C.B. and Williamson, G., Microbiology, vol. 140 (1994), 779-787). The results of the assay are given in Figure 1. As can be deduced from Figure 1, the enzyme of the invention exhibited 50% activity at about pH 5.5 and about 8.5 In order to determine the temperature optimum of the enzyme according to the invention using FAXX as a substrate, FAXX was employed at a concentration of 33 pM and the assay was performed at pH 6.0 in 100 mM MOPS buffer. The temperature of incubation was changed from 20)C to 70)C using a thermostatically controlled spectrophotometer. The release of ferulic acid from FAXX was measured at 335 nm as described above. The results are presented in Figure 2. Kinetics The Km and Vmax of the enzyme of the present invention were determined using FAXX and Ara 2 F (0-[2-0 (trans-feruloyl) -x arabinofuranosyll- (1-5) -L-arabinofuranose) as substrates. FAXX was employed at concentrations varying from 3.72 pM to 49.18 pM and Ara 2 F was used at concentrations ranging from 4.46 FM to 122.92 pAM. The assay was performed at 37 0 C and pH 6.0 in 100 mM MOPS ((3-[N-morpholino]propanesulfonic acid)) buffer with 90 ng enzyme. For both substrates, the release of ferulic acid was measured at 335 nM as described above. Based on results of the above experiments, it was determined that the enzyme of the present invention has the following kinetic constants: substrate Km Vmax FAXX 3.0±0.3 pM 35.6±0.9 pmol/min/mg Ara 2 F 234±27 AM 19.6±1.7 pmol/min/mg.
WO 98/46768 PCT/EP98/02080 12 Hence, the enzyme of the present invention has a Km of about 3. 0 and a Vmax of about 35 when measured under the above conditions using FAXX as a substrate. The specific activity of the enzyme of the present invention was determined for methyl ferulate, methyl coumarate and methyl p-coumarate in an assay at 37 0 C comprising 100 mM MOPS buffer (with 0.02% azide), pH 6.0., 44 ng enzyme and 1 mM of the above substrates. After 15 minutes incubation time, the reaction was terminated by boiling and the free acid liberated was measured using reverse phase HPLC (Kroon, P.A. and Williamson, G., Biotechnol. Appl. Biochem., vol. 23 (1996), 263-267) . The results of the above experiment are shown below. In addition, the specific activity of the enzyme of the present invention was determined for p-nitrophenyl acetate, a naphthyl acetate, a-naphthyl butyrate, a-naphthyl caproate a naphthyl caprylate and a-naphthyl laurate according to the methods described in Ferreira, L.M.A. et al.(Biochem. J., vol. 294 (1993) , 349-355) . The results of the above assay are shown below. substrate specific activity (U*/mg) p-nitrophenyl acetate 204.3 a-naphthyl acetate 121 a-naphthyl butyrate 220 a-naphthyl caproate 256 c-naphthyl caprylate 54 a-naphthyl laurate 6 methyl ferulate 10.6 methyl coumarate 10.5 methyl p-coumarate 2.7 *1 U is defined as the amount of enzyme which gives 1 smol/min of ester hydrolysis.
WO 98/46768 PCT/EP98/02080 13 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Biotechnology and Biological Sciences Research Council (B) STREET: Polaris House, North Star Avenue (C) CITY: Swindon (D) STATE: (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP) : SN2 1UH (ii) TITLE OF INVENTION: Phenolic Acid Esterase and Use Thereof (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 825 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Piromyces equi WO 98/46768 PCT/EP98/02080 14 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..822 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAC AGC GGT CCA ACT GTT GAA TAC TCT ACT GAT GTT GAC TGT TCC GGT 48 Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly 1 5 10 15 AAG ACC CTT AAG AGT AAC ACC AAC CTT AAC ATC AAT GGT CGT AAG GTT 96 Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val 20 25 30 ATT GTA AAA TTC CCA ADC GGC TTC ACT GGT GAC AAG GCT GCT CCA CTT 144 Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu 35 40 45 CTT ATT AAC TAC CAT CCA ATT ATG GGT AGT GCT TCT CAA TGG GAA AGT 192 Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser 50 55 60 GGT TCT CAA ACT GCT AAG GCT GCT TTA AAT GAT GGT GCC ATC GTT GCT 240 Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala 65 70 75 80 TTC ATG GAT GGT GCT CAA GGT CCA ATG GGA CAA GCT TGG AAC GTT GGT 288 Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly 85 90 95 CCA TGT TGT ACT GAT GCT GAT GAT GTT CAA TTC ACT CGT AAC TTC ATT 336 Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile 100 105 110 AAG GAA ATC ACT AGT AAG GCT TOT GTT GAT CCA AAG CGT ATC TAT GCT 384 Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala WO 98/46768 PCT/EP98/02080 15 115 120 125 GCT GGT TTC TCT ATG GGT GGT GGT ATG TCT AAC TAT GCT GGT TGT CAA 432 Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln 130 135 140 CTT GCT GAT GTT ATT GCT GCT GCT GCT CCA TCA GCC TTT GAT CTT GCC 480 Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala 145 150 155 160 AAG GAA ATT GTT GAT GGT GGT AAA TGT AAA CCA GCT CGT CCA TTC CCA 528 Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro 165 170 175 ATC CTT AAC TTC CGT GGT ACT CAA GAT AAC GTT GTT ATG TAC AAC GGT 576 Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly 180 185 190 GGT CTT TCT CAA GTT GTT CAA GGT AAG CCA ATT ACT TTC ATG GGT GCC 624 Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala 195 200 205 AAG AAC AAC TTC AAG GAA TGG GCT AAG ATG AAC GGA TGT ACT GGT GAA 672 Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu 210 215 220 CCA AAA CAA AAC ACT CCA GGT AAC AAC TGT GAA ATG TAC GAA AAC TGT 720 Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys 225 230 235 240 AAG GGT GGT GTT AAG GTT GGT CTT TGC ACT ATC AAC GGT GGT GGT CAC 768 Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His 245 250 255 GCT GAA GGT GAC GGT AAA ATG GGT TGG GAC TTT GTT AAA CAA TTC TCT 816 Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser 260 265 270 WO 98/46768 PCT/EP98/02080 16 CTC CCA TAA 825 Leu Pro (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 274 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly 1 5 10 15 Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val 20 25 30 Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu 35 40 45 Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser 50 55 60 Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala 65 70 75 80 Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly 85 90 95 Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile 100 105 110 WO 98/46768 PCT/EP98/02080 17 Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala 115 120 125 Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln 130 135 140 Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala 145 150 155 160 Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro 165 170 175 Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly 180 185 190 Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala 195 200 205 Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu 210 215 220 Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys 225 230 235 240 Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His 245 250 255 Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser 260 265 270 Leu Pro (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1611 base pairs WO 98/46768 PCT/EP98/02080 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Piromyces equi (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..1608 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG AAG ACT AGC ATT GTA TTA TCT ATC GTT GCT TTA TTT TTA ACA TCC 48 Met Lys Thr Ser Ile Val Leu Ser Ile Val Ala Leu Phe Leu Thr Ser 275 280 285 290 AAA GCT TCT GCT GAT TGT TGG TCA GAA AGA TTA GGT TGG CCA TGC TGT 96 Lys Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys 295 300 305 AGT GAC AGC AAT GCC GAA GTA ATC TAC GTC GAT GAC GAT GGT GAT TGG 144 Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp 310 315 320 GGT GTT GAA AAT AAT GAC TGG TGT GGT ATC CAA AAG GAA GAA GAA AAC 192 Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys Glu Glu Glu Asn 325 330 335 AAT AAC TCA TGG GAT ATG GGT GAT TGG AAC CAA GGT GGT AAC CAA GGT 240 Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly 340 345 350 GGC GGT ATG CCA TGG GGC GAC TTT GGC GGT AAC CAA GGT GGT GGT ATG 288 WO 98/46768 PCT/EP98/02080 19 Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met 355 360 365 370 CAA TGG GGT GAC TTC GGT GGT AAC CAA GGT GGC GGT ATG CCA TGG GGC 336 Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly 375 380 385 GAC TTC GGT GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAC TTT GGC 384 Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly 390 395 400 GGT AAC CAA GGT GGT AAC CAA GGC GGT GGT ATG CCA TGG GGC GAC TTT 432 Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe 405 410 415 GGA GGA AAC CAA GGA GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAT 480 Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp 420 425 430 TTC GGA GGT AAC CAA GGT GGT GGT ATG CAA TGG GGC GAC TTT GGA GGA 528 Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly 435 440 445 450 AAC CAA GGA GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAT TTC GGA 576 Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly 455 460 465 GGT AAC CAA GGT GGT GGT ATG CAA TGG GGC GAC TTT GGA GGA AAC CAA 624 Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln 470 475 480 GGA GGT AAC CAA GGT GGC GGT ATG CCA TGG GGT GAC TTC GGA GGT AAC 672 Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn 485 490 495 CAA GGT GGT GGT ATG CAA TGG GGC GAT TTC GGA GGT AAT CAA GGT GGT 720 Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly WO 98/46768 PCT/EP98/02080 20 500 505 510 GGT ATG CAA TGG GGC GAC TTC GGC GGT AAC CAA GGA GGT AAC CAA GAT 768 Gly Met Gin Trp Gly Asp Phe Gly Gly Asn Gin Gly Gly Asn Gin Asp 515 520 525 530 TGG GGT AAC CAA GGT GGT AAC AGC GGT CCA ACT GTT GAA TAC TCT ACT 816 Trp Gly Asn Gin Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr 535 540 545 GAT GTT GAC TGT TCC GGT AAG ACC CTT AAG AGT AAC ACC AAC CTT AAC 864 Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn 550 555 560 ATC AAT GGT CGT AAG GTT ATT GTA AAA TTC CCA AGC GGC TTC ACT GGT 912 Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr Gly 565 570 575 GAC AAG GCT GCT CCA CTT CTT ATT AAC TAC CAT CCA ATT ATG GGT AGT 960 Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser 580 585 590 GCT TCT CAA TGG GAA AGT GGT TCT CAA ACT GCT AAG GCT GCT TTA AAT 1008 Ala Ser Gin Trp Glu Ser Gly Ser Gin Thr Ala Lys Ala Ala Leu Asn 595 600 605 610 GAT GGT GCC ATC GTT GCT TTC ATG GAT GGT GCT CAA GGT CCA ATG GGA 1056 Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gin Gly Pro Met Gly 615 620 625 CAA GCT TGG AAC GTT GGT CCA TGT TGT ACT GAT GCT GAT GAT GTT CAA 1104 Gin Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gin 630 635 640 TTC ACT CGT AAC TTC ATT AAG GAA ATC ACT AGT AAG GCT TGT GTT GAT 1152 Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp 645 650 655 WO 98/46768 PCT/EP98/02080 21 CCA AAG CGT ATC TAT GCT GCT GGT TTC TCT ATG GGT GGT GGT ATG TCT 1200 Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser 660 665 670 AAC TAT GCT GGT TGT CAA CTT GCT GAT GTT ATT GCT GCT GCT GCT CCA 1248 Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro 675 680 685 690 TCA GCC TTT GAT CTT GCC AAG GAA ATT GTT GAT GGT GGT AAA TGT AAA 1296 Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys 695 700 705 CCA GCT CGT CCA TTC CCA ATC CTT AAC TTC CGT GGT ACT CAA GAT AAC 1344 Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn 710 715 720 GTT GTT ATG TAC AAC GGT GGT CTT TCT CAA GTT GTT CAA GGT AAG CCA 1392 Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro 725 730 735 ATT ACT TTC ATG GGT GCC AAG AAC AAC TTC AAG GAA TGG GCT AAG ATG 1440 Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met 740 745 750 AAC GGA TGT ACT GGT GAA CCA AAA CAA AAC ACT CCA GGT AAC AAC TGT 1488 Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys 755 760 765 770 GAA ATG TAC GAA AAC TGT AAG GGT GGT GTT AAG GTT GGT CTT TGC ACT 1536 Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr 775 780 785 ATC AAC GGT GGT GGT CAC GCT GAA GGT GAC GGT AAA ATG GGT TGG GAC 1584 Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp 790 795 800 WO 98/46768 PCT/EP98/02080 22 TTT GTT AAA CAA TTC TCT CTC CCA TAA 1611 Phe Val Lys Gln Phe Ser Leu Pro 805 810 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 536 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Lys Thr Ser Ile Val Leu Ser Ile Val Ala Leu Phe Leu Thr Ser 1 5 10 15 Lys Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys 20 25 30 Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp 35 40 45 Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys Glu Glu Glu Asn 50 55 60 Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly 65 70 75 80 Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met 85 90 95 Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly 100 105 110 Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly WO 98/46768 PCT/EP98/02080 23 115 120 125 Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe 130 135 140 Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp 145 150 155 160 Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly 165 170 175 Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly 180 185 190 Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln 195 200 205 Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn 210 215 220 Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly 225 230 235 240 Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Asp 245 250 255 Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr 260 265 270 Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn 275 280 285 Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr Gly 290 295 300 Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser 305 310 315 320 WO 98/46768 PCT/EP98/02080 24 Ala Ser Gln Trp Glu Ser Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn 325 330 335 Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly 340 345 350 Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln 355 360 365 Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp 370 375 380 Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser 385 390 395 400 Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro 405 410 415 Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys 420 425 430 Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn 435 440 445 Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro 450 455 460 Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met 465 470 475 480 Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys 485 490 495 Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr 500 505 510 WO 98/46768 PCT/EP98/02080 25 Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp 515 520 525 Phe Val Lys Gln Phe Ser Leu Pro 530 535
Claims (23)
1. Enzyme with phenolic acid esterase activity, characterized in that said enzyme has a pH optimum greater than pH 6.5 and a temperature optimum greater than 45 0 C when measured in a citric acid/disodium hydrogen orthophosphate buffer containing 33 pLM FAXX as a substrate.
2. Enzyme according to claim 1, characterized in that said enzyme has ferulic acid esterase activity and/or coumaric acid esterase activity.
3. Enzyme according to claim 1 or 2, characterized in that said enzyme has a a pH optimum of about pH 7.0 and/or a temperature optimum of about 55 0 C.
4. Enzyme according to any of calims 1 to 3, characterized in that said enzyme is obtainable from Piromyces Sp., preferably Piromyces equi deposited at the International Mycological Institute (IMI) under the accession number
375061.
5. Enzyme according to any of claims 1 to 4, characterized in that said enzyme comprises the amino acid sequence given in SEQ ID NO:1 or SEQ ID NO:3 or functional derivatives thereof.
6. Enzyme according to any of claims 1 to 5, characterized in that said enzyme is encoded by the DNA sequence given in SEQ ID NO:1 or SEQ ID NO:3 or functional derivatives or homologues thereof.
7. DNA molecule encoding an enzyme with phenolic acid esterase activity, characterized in that said DNA molecule comprises a DNA sequence as given in SEQ ID NO:1 WO 98/46768 PCT/EP98/02080 27 or SEQ ID NO:3 or functional derivatives or homologues thereof.
8. DNA molecule encoding an enzyme according to any of claims 1 to 6, characterized in that said DNA molecule comprises a DNA sequence as given in SEQ ID NO:1 or SEQ ID NO:3 or functional derivatives or homologues thereof.
9. DNA molecule according to claim 7 or 8 further comprising vector sequence capable of expressing said enzyme in a procaryotic or eucaryotic host.
10. Transformed procaryotic cell or eucaryotic cell or organism comprising one or more DNA molecules according to claim 7 to 9.
11. Method for the production of an enzyme or enzyme preparation having phenolic acid esterase activity, characterized in that said enzyme is isolated from a cell or organism according to claim 10.
12. Enzyme preparation comprising enzyme according to any of claims 1 to 6 and/or obtainable by the method according to claim 11.
13. Enzyme preparation according to claim 12 comprising one or more further polysaccharide modifying and/or degrading enzymes.
14. Enzyme preparation according to claim 13, characterized in that said polysaccharide modifying and/or degrading enzyme is selected form the group comprising xylanase, arabinanase, a-L-arabinofuranosidase, endoglucanase, a-D glucuronidase, pectinase, acetyl esterase, mannanase, acetyl xylan esterase and other glycosyl hydrolases. WO 98/46768 PCT/EP98/02080 28
15. Enzyme preparation according to any of claims 11 to 14 comprising one or more further enzymes selected from the group comprising amylase, protease, a-galactosidase, phytase and lipase.
16. Use of the enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15 in a process for releasing or preparing phenolic acids from a substrate comprising phenolic acid moieties.
17. Use of the enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15 in the production of animal feed.
18. Use of the enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15 in the production of food.
19. Use of the enzyme enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15 in the production of paper.
20. Use of the enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15 in a process for bioconversion of plant material or ligno-cellulose wastes to sugars.
21. Use of the enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15 in crop plants to improve the digestibility of said plants for livestock.
22. Feed additive comprising the enzyme according to any of claims 1 to 6 and/or enzyme preparation according to any of claims 11 to 15.
23. Feed comprising the feed additive according to claim 22.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9707540A GB2324302A (en) | 1997-04-14 | 1997-04-14 | Phenolic acid esterase |
| GB9707540 | 1997-04-14 | ||
| PCT/EP1998/002080 WO1998046768A2 (en) | 1997-04-14 | 1998-04-09 | Phenolic acid esterase and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU7642398A true AU7642398A (en) | 1998-11-11 |
Family
ID=10810766
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU76423/98A Abandoned AU7642398A (en) | 1997-04-14 | 1998-04-09 | Phenolic acid esterase and use thereof |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0975768A2 (en) |
| JP (1) | JP2001523090A (en) |
| CN (1) | CN1255165A (en) |
| AU (1) | AU7642398A (en) |
| BR (1) | BR9808554A (en) |
| CA (1) | CA2286694A1 (en) |
| GB (1) | GB2324302A (en) |
| WO (1) | WO1998046768A2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6365390B1 (en) | 1998-09-04 | 2002-04-02 | University Of Georgia Research Foundation, Inc. | Phenolic acid esterases, coding sequences and methods |
| US6602700B1 (en) | 1998-09-04 | 2003-08-05 | University Of Georgia Research Foundation, Inc. | Phenolic acid esterases, coding sequences and methods |
| GB0216784D0 (en) * | 2002-07-18 | 2002-08-28 | Biocatalysts Ltd | Feruloyl esterase and uses thereof |
| WO2006081825A1 (en) * | 2005-02-04 | 2006-08-10 | University Of Aarhus | A method for recycling important nutritional elements from waste |
| GB0716702D0 (en) * | 2007-08-28 | 2007-10-10 | Biocatalysts Ltd | Enzyme productions |
| EP2411511B1 (en) * | 2009-03-24 | 2018-08-08 | Novozymes A/S | Polypeptides having acetyl xylan esterase activity and polynucleotides encoding same |
| CN102220298A (en) * | 2011-04-20 | 2011-10-19 | 中国科学院微生物研究所 | Ferulic acid esterase FaeI as well as coding gene and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2301103B (en) * | 1995-05-23 | 1999-12-22 | Danisco | An enzyme system comprising ferulic acid esterase |
-
1997
- 1997-04-14 GB GB9707540A patent/GB2324302A/en not_active Withdrawn
-
1998
- 1998-04-09 CN CN98804186A patent/CN1255165A/en active Pending
- 1998-04-09 EP EP98924104A patent/EP0975768A2/en not_active Withdrawn
- 1998-04-09 JP JP54346098A patent/JP2001523090A/en active Pending
- 1998-04-09 WO PCT/EP1998/002080 patent/WO1998046768A2/en not_active Application Discontinuation
- 1998-04-09 AU AU76423/98A patent/AU7642398A/en not_active Abandoned
- 1998-04-09 BR BR9808554-9A patent/BR9808554A/en not_active IP Right Cessation
- 1998-04-09 CA CA002286694A patent/CA2286694A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| GB9707540D0 (en) | 1997-06-04 |
| CA2286694A1 (en) | 1998-10-22 |
| GB2324302A (en) | 1998-10-21 |
| WO1998046768A3 (en) | 1999-01-21 |
| EP0975768A2 (en) | 2000-02-02 |
| WO1998046768A2 (en) | 1998-10-22 |
| BR9808554A (en) | 2000-05-23 |
| CN1255165A (en) | 2000-05-31 |
| JP2001523090A (en) | 2001-11-20 |
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