CA2286694A1 - Phenolic acid esterase and use thereof - Google Patents
Phenolic acid esterase and use thereof Download PDFInfo
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Abstract
The present invention relates to an enzyme with phenolic acid esterase activity, gene encoding said enzyme as well as a method for the production and use of said enzyme.
Description
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 stability 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 (3-(1-4)-D-xylopyranose pol~,nner which can be substituted with sugar residues, including a-L-arabinose, and c~.-D-glucuronic acid and/or the 4-0-methyl ether derivative of cc-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).
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 stability 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 (3-(1-4)-D-xylopyranose pol~,nner which can be substituted with sugar residues, including a-L-arabinose, and c~.-D-glucuronic acid and/or the 4-0-methyl ether derivative of cc-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).
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 enzymes) 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-O-[(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-coumaroyl]-a-L-arabinofuranosyl}-(1-3)-O-(i-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°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°C.
WO 98/46768 PCT/EP98/02080 ..
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-O-[(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-coumaroyl]-a-L-arabinofuranosyl}-(1-3)-O-(i-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°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°C.
WO 98/46768 PCT/EP98/02080 ..
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°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 t:o provide a method for the production of an enzyme with pher~olic 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°C, preferably greater than 50°C, and more preferably about 55°C, when measured in a citric acid/disodium hydrogen orthophosphate buffer containing 33 ~,M FAXX as a substrate. Preferably, said enzyme has ferulic acid esterase activity and coumaric acid esterase activity.
WO 98/46768 PCT/EP98/02080 ..
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 t:o provide a method for the production of an enzyme with pher~olic 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°C, preferably greater than 50°C, and more preferably about 55°C, when measured in a citric acid/disodium hydrogen orthophosphate buffer containing 33 ~,M FAXX as a substrate. Preferably, said enzyme has ferulic acid esterase activity and coumaric acid esterase activity.
WO 98/46768 PCT/EP98/02080 ..
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), insertions) and/or deletions) 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°C, preferably greater than 50°C, and more preferably about 55°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 N0:3 or a functional derivative thereof.
In addition, the enzyme of the present invention is preferably encoded by the DNA sequence given in SEQ ID N0:1 or a functional derivative thereof. More preferably, the enzyme of the present invention is encoded by the DNA sequence given in SEQ ID N0:3 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 N0:1 or a functional derivative or homologue thereof. A functional derivative of the DNA sequence given in SEQ ID N0:1 is defined as a DNA
. CA 02286694 1999-10-13 sequence having one or more 5'-, 3'- or internal substitution(s), insertions) and/or deletions) 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°C, preferably greater than 50°C, and more preferably about 55°C, when measured in a citric acid/disodium hydrogen orthophosphate buffer comprising 33 uM
FAXX as a substrate. A functional homologue of the DNA
sequence of the present invention is defined as a DNA sequence with preferably 75o homology, more preferably 85o homology and most preferably 95o homology to the DNA sequence given in SEQ
ID NO:1 or SEQ~ID N0:3. More preferably, a DNA molecule encoding an enzyme according to the present invention comprises a DNA sequence as given in SEQ ID N0:3 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.
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 enzymes) is (are) preferably selected from the group comprising xylanase, arabinanase, a,-L-arabinofuranosidase, endoglucanase, a-D-glucuronidase, pectinase, acetyl esterase, mannanase, acetyl xylan esterase and other glycosyl hydrolases.
In addition, the enzyme preparation of the present inventio:~
can also include one or more further enzymes selected from the group comprising amylase, protease, a-galactosidase, phytase and lipase.
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 phenoiic 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 witrF 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.
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°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 0.100 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 ~,ZAPII 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.
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 picoBlueT''t immunoscreeni.ng kit (Stratagene) , with the following modifications: isopropyl-~i-D-thiogalactopyranoside (IPTG: 0.33 mM) was added directly to the soft agar overlays containing recombinant a.ZAPII and host bacteria (E. coli XL1-Blue); plaques were lifted onto Hybond-C filters (Amersham);
blocking solution contained dried milk powder (4o 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 ~.l/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 w having phenolic acid esterase activity of the present invention was performed and the results are given in SEQ ID
N0:3. The open reading frame comprises 1608 nucleotides, 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°C, 45 seconds at SO°C, and 1 minute at 72°C) in a buffer comprising 50 mM
Tris-HCl, pH 9.0, 50 mM NaCl, 10 mM MgCl2, 200 ~.M 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°C with a final FAXX concentration of 33 ACM. Ferulic acid release from FAXX was monitored continuously for 3 min at 335 nm according to (Faulds, C.B.
and Williamson, G., Microbiology, ~rol. 140 (1994), 779-787).
The results of the assay are give=_n in Figure 1. As can be deduced from Figure 1, the enzyme of the invention exhibited 50o activity at about pH 5.5 and a:oout 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 ~.M 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 Umax of the enzyme of. the present invention were determined using FAXX and ArazF (0-(2-O (trans-feruloyl) -a.-arabinofuranosyl]-(1-5)-L-arabinofuranose) as substrates. FAXX
was employed at concentrations varying from 3.72 ~,M to 49.18 ~,M and Ara2F was used at concentrations ranging from 4.46 ~,M
to 122.92 ~M. The assay was performed at 37°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 Umax FAXX 3.0~0.3 ~.M 35.6~0.9 umol/min/mg Ara2F 234~27 ~.M 19.6~1.7 ~,mol/min/mg.
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°C comprising 100 mM MOPS
buffer (with 0.020 azide), pH 6Ø, 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 cc-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 a-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 ~,mol/min of ester hydrolysis.
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 lUfi (ii) TITLE OF INVENTION: Phenolic Acid Esterase and Us2 ThereoL
(iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/INS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID N0: 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 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..822 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asn Ser G1y Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly A.~G 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 G1y Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu Leu I1e Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser 5p 55 60 Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala IS
Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys G1n Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro ile Leu Asn Phe Arg Gly Thr Gln Asp Asr, Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met. Asn Gly Cys Thr Gly Glu CCA AAA CAA AAC ACT CCA GGT AAC AAC TG'r GAA ATG TAC GAA AAC TGT 720 Pro Lys Gln Asn Thr Pro Gly Asn Asn Cya Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser WO 98/46768 PCT/EP98/02080 .
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 N0: 2:
Asn Ser Gly Pro Thr Va1 Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro ;per Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly G1y Lys Cys Lys Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser Leu Pro (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1611 base pairs (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 N0: 3:
Met Lys Thr Ser Ile Val Leu Ser Ile Val Ala Leu Phe Leu Thr Ser Lys Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys Glu Glu Glu Asn Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Cily Gly Met Pro Trp Gly Aso Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly 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 Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly G1y Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn 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 ..
Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Asp Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser G1y Phe Thr Gly Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu 5er Gly Ser G1n Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala A1a Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro ATT ACT TTC ATG GGT GCC AAG AAC AAC TTC.' AAG GAA TGG GCT AAG ATG 1440 Ile Thr Ph2 Met Gly Ala Lys Asn Asn Phe: Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys GAA ATG TAC GAA AAC TGT AAG GGT GGT GT'T AAG GTT GGT CTT TGC ACT 1536 Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser Leu Pro (2) INFORMATION FOR SEQ ID N0: 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 Lvs Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys G1u Glu Glu Asn Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met G1n Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly 165 1.70 175 Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly G1y Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met G1n Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn G1n Asp Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr G1y Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala G1y Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser A1a Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser Leu Pro
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), insertions) and/or deletions) 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°C, preferably greater than 50°C, and more preferably about 55°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 N0:3 or a functional derivative thereof.
In addition, the enzyme of the present invention is preferably encoded by the DNA sequence given in SEQ ID N0:1 or a functional derivative thereof. More preferably, the enzyme of the present invention is encoded by the DNA sequence given in SEQ ID N0:3 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 N0:1 or a functional derivative or homologue thereof. A functional derivative of the DNA sequence given in SEQ ID N0:1 is defined as a DNA
. CA 02286694 1999-10-13 sequence having one or more 5'-, 3'- or internal substitution(s), insertions) and/or deletions) 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°C, preferably greater than 50°C, and more preferably about 55°C, when measured in a citric acid/disodium hydrogen orthophosphate buffer comprising 33 uM
FAXX as a substrate. A functional homologue of the DNA
sequence of the present invention is defined as a DNA sequence with preferably 75o homology, more preferably 85o homology and most preferably 95o homology to the DNA sequence given in SEQ
ID NO:1 or SEQ~ID N0:3. More preferably, a DNA molecule encoding an enzyme according to the present invention comprises a DNA sequence as given in SEQ ID N0:3 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.
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 enzymes) is (are) preferably selected from the group comprising xylanase, arabinanase, a,-L-arabinofuranosidase, endoglucanase, a-D-glucuronidase, pectinase, acetyl esterase, mannanase, acetyl xylan esterase and other glycosyl hydrolases.
In addition, the enzyme preparation of the present inventio:~
can also include one or more further enzymes selected from the group comprising amylase, protease, a-galactosidase, phytase and lipase.
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 phenoiic 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 witrF 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.
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°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 0.100 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 ~,ZAPII 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.
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 picoBlueT''t immunoscreeni.ng kit (Stratagene) , with the following modifications: isopropyl-~i-D-thiogalactopyranoside (IPTG: 0.33 mM) was added directly to the soft agar overlays containing recombinant a.ZAPII and host bacteria (E. coli XL1-Blue); plaques were lifted onto Hybond-C filters (Amersham);
blocking solution contained dried milk powder (4o 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 ~.l/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 w having phenolic acid esterase activity of the present invention was performed and the results are given in SEQ ID
N0:3. The open reading frame comprises 1608 nucleotides, 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°C, 45 seconds at SO°C, and 1 minute at 72°C) in a buffer comprising 50 mM
Tris-HCl, pH 9.0, 50 mM NaCl, 10 mM MgCl2, 200 ~.M 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°C with a final FAXX concentration of 33 ACM. Ferulic acid release from FAXX was monitored continuously for 3 min at 335 nm according to (Faulds, C.B.
and Williamson, G., Microbiology, ~rol. 140 (1994), 779-787).
The results of the assay are give=_n in Figure 1. As can be deduced from Figure 1, the enzyme of the invention exhibited 50o activity at about pH 5.5 and a:oout 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 ~.M 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 Umax of the enzyme of. the present invention were determined using FAXX and ArazF (0-(2-O (trans-feruloyl) -a.-arabinofuranosyl]-(1-5)-L-arabinofuranose) as substrates. FAXX
was employed at concentrations varying from 3.72 ~,M to 49.18 ~,M and Ara2F was used at concentrations ranging from 4.46 ~,M
to 122.92 ~M. The assay was performed at 37°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 Umax FAXX 3.0~0.3 ~.M 35.6~0.9 umol/min/mg Ara2F 234~27 ~.M 19.6~1.7 ~,mol/min/mg.
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°C comprising 100 mM MOPS
buffer (with 0.020 azide), pH 6Ø, 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 cc-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 a-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 ~,mol/min of ester hydrolysis.
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 lUfi (ii) TITLE OF INVENTION: Phenolic Acid Esterase and Us2 ThereoL
(iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/INS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID N0: 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 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..822 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asn Ser G1y Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly A.~G 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 G1y Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu Leu I1e Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser 5p 55 60 Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala IS
Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys G1n Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro ile Leu Asn Phe Arg Gly Thr Gln Asp Asr, Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met. Asn Gly Cys Thr Gly Glu CCA AAA CAA AAC ACT CCA GGT AAC AAC TG'r GAA ATG TAC GAA AAC TGT 720 Pro Lys Gln Asn Thr Pro Gly Asn Asn Cya Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser WO 98/46768 PCT/EP98/02080 .
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 N0: 2:
Asn Ser Gly Pro Thr Va1 Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr Gly Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro ;per Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly G1y Lys Cys Lys Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser Leu Pro (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1611 base pairs (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 N0: 3:
Met Lys Thr Ser Ile Val Leu Ser Ile Val Ala Leu Phe Leu Thr Ser Lys Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys Glu Glu Glu Asn Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Cily Gly Met Pro Trp Gly Aso Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly 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 Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly G1y Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn 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 ..
Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Asp Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser G1y Phe Thr Gly Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu 5er Gly Ser G1n Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala A1a Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala Gly Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser Ala Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro ATT ACT TTC ATG GGT GCC AAG AAC AAC TTC.' AAG GAA TGG GCT AAG ATG 1440 Ile Thr Ph2 Met Gly Ala Lys Asn Asn Phe: Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys GAA ATG TAC GAA AAC TGT AAG GGT GGT GT'T AAG GTT GGT CTT TGC ACT 1536 Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser Leu Pro (2) INFORMATION FOR SEQ ID N0: 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 Lvs Ala Ser Ala Asp Cys Trp Ser Glu Arg Leu Gly Trp Pro Cys Cys Ser Asp Ser Asn Ala Glu Val Ile Tyr Val Asp Asp Asp Gly Asp Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Ile Gln Lys G1u Glu Glu Asn Asn Asn Ser Trp Asp Met Gly Asp Trp Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met G1n Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly 165 1.70 175 Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly G1y Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn Gln Gly Gly Gly Met Pro Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met G1n Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Gly Met Gln Trp Gly Asp Phe Gly Gly Asn Gln Gly Gly Asn G1n Asp Trp Gly Asn Gln Gly Gly Asn Ser Gly Pro Thr Val Glu Tyr Ser Thr Asp Val Asp Cys Ser Gly Lys Thr Leu Lys Ser Asn Thr Asn Leu Asn Ile Asn Gly Arg Lys Val Ile Val Lys Phe Pro Ser Gly Phe Thr G1y Asp Lys Ala Ala Pro Leu Leu Ile Asn Tyr His Pro Ile Met Gly Ser Ala Ser Gln Trp Glu Ser Gly Ser Gln Thr Ala Lys Ala Ala Leu Asn Asp Gly Ala Ile Val Ala Phe Met Asp Gly Ala Gln Gly Pro Met Gly Gln Ala Trp Asn Val Gly Pro Cys Cys Thr Asp Ala Asp Asp Val Gln Phe Thr Arg Asn Phe Ile Lys Glu Ile Thr Ser Lys Ala Cys Val Asp Pro Lys Arg Ile Tyr Ala Ala Gly Phe Ser Met Gly Gly Gly Met Ser Asn Tyr Ala G1y Cys Gln Leu Ala Asp Val Ile Ala Ala Ala Ala Pro Ser A1a Phe Asp Leu Ala Lys Glu Ile Val Asp Gly Gly Lys Cys Lys Pro Ala Arg Pro Phe Pro Ile Leu Asn Phe Arg Gly Thr Gln Asp Asn Val Val Met Tyr Asn Gly Gly Leu Ser Gln Val Val Gln Gly Lys Pro Ile Thr Phe Met Gly Ala Lys Asn Asn Phe Lys Glu Trp Ala Lys Met Asn Gly Cys Thr Gly Glu Pro Lys Gln Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys Lys Gly Gly Val Lys Val Gly Leu Cys Thr Ile Asn Gly Gly Gly His Ala Glu Gly Asp Gly Lys Met Gly Trp Asp Phe Val Lys Gln Phe Ser Leu Pro
Claims (21)
1. Enzyme with phenolic acid esterase activity including ferulic acid esterase activity and coumaric acid esterase activity, characterized in that said enzyme has a pH optimum greater than pH 6.5 and a temperature optimum greater than 45°C when measured in a citric acid/disodium hydrogen orthophosphate buffer containing 33 µM FAXX as a substrate as well as a Km of about 3.0µm and a Vmax of about 35µmol/min/mg protein when measured at 37°C and pH 6.0 in MOPS buffer containing FAXX as a substrate.
2. Enzyme according to claim l, characterized in that said enzyme has a pH optimum of about pH 7.0 and/or a temperature optimum of about: 55°C.
3. Enzyme according to any of claims 1 or 2, 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.
4. Enzyme according to any of claims 1 to 3, 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.
5. Enzyme according to any of claims 1 to 4, 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.
6. DNA molecule encoding an enzyme according to any of claims 1 to 5, 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.
ID NO:3 or functional derivatives or homologues thereof.
7. DNA molecule according to claim 6 further comprising vector sequence capable of expressing said enzyme in a procaryotic or eucaryotic host.
8. Transformed procaryotic cell or eucaryotic cell or organism comprising one or more DNA molecules according to claim 6 or 7.
9. Method for the production of an enzyme or enzyme preparation having phenolic acid esterase activity according to any of claims 1 to 5, characterized in that said enzyme is isolated from a cell or organism according to claim 8.
10. Enzyme preparation comprising enzyme according to any of claims 1 to 5 and/or obtainable by the method according to claim 9.
11. Enzyme preparation according to claim 10 comprising one or more further polysaccharide modifying and/or degrading enzymes.
12. Enzyme preparation according to claim 11, characterized in that said polysaccharide modifying and/or degrading enzyme is selected from the group comprising xylanase, .alpha.-arabinanase, .alpha.-L-arabinofuranosidase, endoglucanase, D-glucuronidase, pectinase, acetyl esterase, mannanase, acetyl xylan esterase and other glycosyl hydrolases.
13. Enzyme preparation according to any of claims 10 to 12 comprising one or more further enzymes selected from the group comprising amylase, protease, .alpha.-galactosidase, phytase and lipase.
14. Use of the enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13 in a process for releasing or preparing phenolic acids from a substrate comprising phenolic acid moieties.
15. Use of the enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13 in the production of animal feed.
16. Use of the enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13 in the production of food.
17. Use of the enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13 in the production of piper.
18. Use of the enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13 in a process for bioconversion of plant material or ligno-cellulose wastes to sugars.
19. Use of the enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13 in crop plants to improve the digestibility of said plants for livestock.
20. Feed additive comprising tree enzyme according to any of claims 1 to 5 and/or enzyme preparation according to any of claims 10 to 13.
21. Feed comprising the feed additive according to claim 20.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9707540.2 | 1997-04-14 | ||
| GB9707540A GB2324302A (en) | 1997-04-14 | 1997-04-14 | Phenolic acid esterase |
| PCT/EP1998/002080 WO1998046768A2 (en) | 1997-04-14 | 1998-04-09 | Phenolic acid esterase and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2286694A1 true CA2286694A1 (en) | 1998-10-22 |
Family
ID=10810766
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002286694A Abandoned CA2286694A1 (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 |
|---|---|---|---|---|
| AU754791B2 (en) * | 1998-09-04 | 2002-11-28 | University Of Georgia Research Foundation, Inc., The | 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 |
| EP1853534A1 (en) * | 2005-02-04 | 2007-11-14 | University of Aarhus | A method for recycling important nutritional elements from waste |
| GB0716702D0 (en) * | 2007-08-28 | 2007-10-10 | Biocatalysts Ltd | Enzyme productions |
| BRPI1013483B1 (en) * | 2009-03-24 | 2019-04-24 | Novozymes A/S | NUCLEIC ACID CONSTRUCTION, RECOMBINANT EXPRESSION VECTOR, TRANSGENIC MICROBIAN HOST CELL, METHODS FOR PRODUCING POLYPEPTIDE, TO DEGRADE ACTILED XYLAN AND TO PRODUCE COMPOSING PRODUCT, AND FERMENTATION PRODUCT, |
| 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 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 JP JP54346098A patent/JP2001523090A/en active Pending
- 1998-04-09 CA CA002286694A patent/CA2286694A1/en not_active Abandoned
- 1998-04-09 WO PCT/EP1998/002080 patent/WO1998046768A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JP2001523090A (en) | 2001-11-20 |
| GB2324302A (en) | 1998-10-21 |
| WO1998046768A3 (en) | 1999-01-21 |
| CN1255165A (en) | 2000-05-31 |
| WO1998046768A2 (en) | 1998-10-22 |
| GB9707540D0 (en) | 1997-06-04 |
| EP0975768A2 (en) | 2000-02-02 |
| BR9808554A (en) | 2000-05-23 |
| AU7642398A (en) | 1998-11-11 |
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| FZDE | Discontinued |