Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • 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

Definitions

• 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.
• 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.
• xylans He icelluloses 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 ⁇ - (1-4) -D-xylopyranose polymer which can be substituted with sugar residues, including ⁇ -L-arabinose, and ⁇ -D- glucuronic acid and/or the 4-0-methyl ether derivative of ⁇ -D- glucuronic acid.
• Many xylans are also esterified with phenolic acid residues, including coumaric acid and ferulic acid.
• phenolic acid residues are present in an ester linkage to ⁇ -L-arabinofuranosyl xylan and can serve to protect xylan from xylan-degrading enzymes, so-called xylanase ⁇ , as well they confer structural stability to the plant cell wall by forming covalent bonds with the lignin present therein.
• 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) .
• microorganisms 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.
• FAE-II was reported to be specific for the substrate (0- ⁇ 5-0- [ (E) -feruloyl] - ⁇ -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] - ⁇ -L-arabinofuranosyl ⁇ - (1-3) -O- ⁇ -D-xylopyranosyl- (1- 4) -D- ylopyranose (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.
• 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.
• 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.
• said enzyme has ferulic acid esterase activity and coumaric acid esterase activity.
• Subject matter of the present invention is also an enzyme with phenolic acid esterase activity obtainable from Piromyces Sp .
• Piromyces equi 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.
• IMI International Mycological Institute
• 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- erminal, 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°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 NO : 3 or a functional derivative thereof.
• 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.
• 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:l 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 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°C, preferably greater than 50°C, and more preferably about 55°C, when measured in a citric acid/disodium hydrogen orthophosphate buffer comprising 33 ⁇ M 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:l 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 .
• 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 .
• a transformed procaryotic cell or eucaryotic cell comprising one or more DNA molecules of the present invention.
• said cells are selected from the group comprising E. coli, Bacillus sp . , such as Bacillus subtilis, Lactobacillus ⁇ p . , 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 .
• transgenic plants such as maize, soybean and canola/rapeseed.
• 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.
• Transgenic fungus such as Aspergillus
• tranformed yeast such as Saccharomyces
• 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, ⁇ -L-arabinofuranosidase, endoglucanase, ⁇ -D-glucuronidase, pectinase, acetyl esterase, mannanase, acetyl xylan esterase and other glycosyl hydrolases .
• the enzyme preparation of the present invention can also include one or more further enzymes selected from the group comprising amylase, protease, ⁇ -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.
• 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.
• 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.
• the enzy e 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 .
• Figure 1 pH profile of the phenolic acid esterase of the present invention measured using FAXX as a substrate .
• FIG. 1 Temperature profile of the phenolic acid esterase of the present invention measured using FAXX as a substrate.
• 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.
• Recombinant phage were grown by plating on lawns of E. coli XLl-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) .
• 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.
• 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, encoding a protein of 536 amino acids with a predicted molecular weight of 55,540 daltons .
• a truncated enzyme encoded by SEQ ID NO . 1 was generated in a
• 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 Mcllvaine'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.
• Mcllvaine'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
• the assay was carried out at 37 °C with a final FAXX concentration of 33 ⁇ M. Ferulic acid release from FAXX was monitored continuously for 3 min at 335 n according to (Faulds, C.B. and Williamson, G., Microbiology, vol. 140 (1994), 779-787).
• 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.
• the K m and V max of the enzyme of the present invention were determined using FAXX and Ara 2 F (0-[2-0 (trans-feruloyl) - ⁇ - arabinofuranosyl]- (1-5) -L-arabinofuranose) as substrates.
• FAXX was employed at concentrations varying from 3.72 ⁇ M to 49.18 ⁇ M and Ara 2 F 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.
• the enzyme of the present invention has a K m of about 3.0 and a V max 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 0.02% 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 .
• the specific activity of the enzyme of the present invention was determined for p-nitrophenyl acetate, ⁇ - naphthyl acetate, -naphthyl butyrate, ⁇ -naphthyl caproate ⁇ - naphthyl caprylate and ⁇ -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.
• CCA AAA CAA AAC ACT CCA GGT AAC AAC TGT GAA ATG TAC GAA AAC TGT 720 Pro Lys Gin Asn Thr Pro Gly Asn Asn Cys Glu Met Tyr Glu Asn Cys 225 230 235 240
• MOLECULE TYPE DNA (genomic)
• GAT GGT GCC ATC GTT GCT TTC ATG GAT GGT GCT CAA GGT CCA ATG GGA 1056 Asp Gly Ala He Val Ala Phe Met Asp Gly Ala Gin Gly Pro Met Gly 615 620 625

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• 1997
  • 1997-04-14 GB GB9707540A patent/GB2324302A/en not_active Withdrawn
• 1998
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