MXPA99003051A - Esterases, dna encoding therefor and vectors and host cells incorporating same - Google Patents

Abstract

A novel DNA is provided which encodes an enzyme having esterolytic activity isolated from Aspergillus. Also provided for is a method of isolating DNA encoding an enzyme having esterolytic activity from organisms which possess such DNA, transformation of the DNA into a suitable host organism, expression of the transformed DNA and the use of the expressed esterase protein in feed as a supplement, in textiles for the finishing of such textiles prior to sale, in starch processing or production of foods such as baked bread.

Description

ESTERASAS, DNA THAT CODIFIES FOR THEMSELVES AND VECTORS AND GUEST CELLS THAT INCORPORATE THE SAME BACKGROUND OF THE INVENTION The present invention is directed to a novel sterolitic enzyme, to the new genetic material coding for this enzyme and to the sterolitic proteins developed from it. In particular, the present invention provides an esterase derived from Aspergillus, a DNA encoding that esterase, the vectors comprising that DNA, the host cells transformed with that DNA and a protein product produced by such host cells. Xylan, together with cellulose, is the most abundant renewable polysaccharide in nature. This is the largest hemicellulosic component in plants and is located predominantly in the secondary cell walls of angiosperms and gymnosperms. The composition and structure of xylan are more complicated than those of cellulose, and can vary quantitatively and qualitatively in various woody plant species, grasses and cereals. He REF .: 29978 Xylan is a heteropolymer in which the constituents are linked together not only with glycosidic bonds but also by ester bonds. Ferulic acid is the most abundant hydroxycinnamic acid found in plants, and is known to be esterified to arabinose in wheat bran, wheat flour, barley straw, corn, sugarcane bagasse, rice straw, and other monocotyledons, and this is also found in galactose residues in the pectin of sugar beet, spinach, and other dicots. P-coumaric acid is also bound in a similar way in monocotyledons. The presence of other phenolic acids has been shown to limit the biodegradation of the cell wall and play significant roles in the extension of the cell wall and its stabilization through the crosslinking of heteroxylan chains by the formation of phenolic dimers via the peroxidases and / or the photodimerization initiated by sunlight. In addition, phenolic acids have been shown to function as crosslinkers between the cell wall polysaccharides and the phenylpropanoid lignin polymer. The coupling or covalent bond from lignin to wall polysaccharides and crosslinking of xylan chains within hemicellulose limit the total bioavailability of the polysaccharide, resulting in significant amounts of undigested fiber in animal feeds, poor bioconversion of agricultural residue in useful products and incomplete processing of grains. The enzymatic hydrolysis of xylan to its monomers requires the participation of several enzymes with different functions. These are classified into two groups based on the nature of the links they break. The first group of enzymes is the hydrolases (EC 3.2.1) involved in the hydrolysis of the glycosidic bonds of xylan. These include endo-xylanases (EC-3.2.1.8) which randomly dismantle the xylan backbone in shorter xylooligosaccharides; β-xylosidase (EC 3.2.1.37), which breaks the xylooligosaccharides in an exo manner, producing xylose, α-L-arabinofuranosidase (EC.3.2.1.55), and a-glucuronidase (EC 3.2.1.1) which are withdrawn. the arabinose and the 4-O-methylglucuronic acid substituents, respectively, of the xylan backbone. The second group includes enzymes that hydrolyze the ester linkages (esterase, EC 3.1.1) between the xylose units and the xylan polymer and the acetyl groups (acetyl xylan esterase, EC 3.1.1.6) or between the arabinosyl groups and phenolic portions such as ferulic acid (feruloyl esterase) and p-coumaric acid (coumaroyl esterase). Faulds et al. Reported two forms of ferulic acid esterase isolated from Aspergill us niger. The different esterases are distinguished based on the molecular weight and the specificity of the substrate (Faulds et al.

Biotech, Appl. Biochem., Vol 17, pp. 349-359 (1993)). Brezillon et al. Reported the existence of at least two cinnamoyl esterases, which were believed to be different from the ferulic acid esterases shown in the prior art.

(Brezillon et al., Appl. Microb.

Biotechnol., Vol. 45, pp. 371-376 (1996)). An esterase of ferulic acid called FAE-III was isolated from Aspergillus ni ger CBS 120.49 and showed that it acts together with xylanase to eliminate almost all ferulic acid and low molecular mass xylooligosaccharides in a preparation of wheat bran; Ferulic acid was also eliminated without the addition of xylanase, although at a lower level. Faulds et al. Also isolated and partially characterized Aspergillus FAE-III or CBS120.49 gel developed in spelled xylene (Faulds et al., Microbiology, vol.140, pp. 779-787 (1994)) and showed that it had a pl of 3.3, a molecular weight of 36kD (SDS-PAGE) and 14.5 kD (Gel Filtration method), an optimum pH of 5 and an optimum temperature of 55-60 ° C; The bond of microcrystalline cellulose was also detected. The authors launched the theory that FAE-II can be a proteolytically modified FAE-III. Recently, the different esterases of ferulic acid known, derived from Aspergi llus niger, have been distinguished with base in their different substrate specificity and it was noted that FAE-II and FAE-III were incapable of releasing ferulic acid from the pulp. of sugar beet (Brezillon et al., supra). However, in spite of the characterization work that has been directed to the Aspergill us niger esterases, a need remains in the art for additional esterases for their various applications. In addition, those skilled in the art have thus failed in discover a nucleotide sequence that can be used to produce more efficient organisms manipulated by genetic engineering, capable of expressing such esterases in large quantities, suitable for industrial production. However, there is a pressing need for the development of an esterase expression system via genetic engineering, which makes possible the purification and use of relatively pure enzyme-work quantities.

BRIEF DESCRIPTION OF THE INVENTION An object of the present invention is to provide the new esterase proteins and the DNA encoding such proteins. It is an object of the present invention to provide a method of isolating DNA from many different species, whose DNA codes for the protein having esterase activity. A further objective of the present invention is to provide an esterase that is produced by a cell. adequate guest, which has been transformed by the DNA that encodes the sterolitic activity. The present invention provides a purified 38 kD esterase which is derived from Aspergillus niger. In addition, a DNA sequence encoding 38kD esterase is provided which comprises a DNA as shown in Figure 5 (SEQ ID No. 27); a DNA encoding the amino acid sequence is also shown in Figure 5 (SEQ ID No. 28); a DNA encoding an esterase comprising an amino acid segment that differs in sequence in Figure 5, with the proviso that the DNA codes for a 38 kD esterase derivative specifically described therein; and a DNA encoding an esterase comprising an amino acid segment that differs from the sequence in Figure 5, with the proviso that the DNA hydrolyzes low demand conditions and / or standard requirement conditions, as defined below, with a DNA comprising all or part of the DNA in Figure 5. The present invention further encompasses vectors that include the DNA sequences described above, host cells that have been transformed with such DNA or vectors, the charges of fermentation comprising such host cells and the esterase proteins encoded by such DNA, which are expressed by the host cells. Preferably, the DNA of the invention is a substantially purified form and is used to prepare a transformed host cell, capable of producing the protein product encoded therein. In addition, polypeptides that are products of expression of the DNA sequences described above are within the scope of the present invention. The enzyme of the present invention has application as a supplement for an animal feed; in a process for the treatment of cloth; to improve the mechanical properties of the dough and the final product of baking food; in the modification of polysaccharides to give novel properties; for example, gums; and in the processing of grains. In addition, the enzyme also has application in the processing of plant materials for the release of phenolic groups for use as an oxidizing, photoprotective, anti-inflammatory and / or antimicrobial agent, which find use in personal care products, such as cosmetics and as an assistant in the conversion of chemical food materials to specialty chemicals, valuable, food additives and flavorings. An advantage of the present invention is that a DNA has been isolated which provides the ability to isolate additional DNAs, which code for the protein having sterolic activity. Another advantage of the present invention is that, by virtue of the provision of a DNA encoding a protein having sterolitic activity, it is possible to produce, via recombinant means, a host cell that is capable of producing a protein having sterolitic activity in relatively large quantities. Yet another advantage of the present invention is that commercial application of proteins having sterolitic activity becomes practical.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a western blot after the SDS-PAGE gel showing the fragmentation of FAE under denaturing conditions.

Figure 2 illustrates the DNA sequence (SEQ ID No. 25) with the introns and amino acid sequence (SEQ ID No. 26) of a 650 base pair fragment corresponding to the gene coding for a 50 kD esterase isolated from Aspergi l l us ni ni ger.

Figure 3 illustrates the band corresponding to the 38 kD esterase after purification.

Figure 4 illustrates a restriction map of a DNA fragment containing the gene coding for the 38 kD esterase.

Figure 5 illustrates the complete DNA (SEQ ID No. 27) with enhancement for signaling the signal sequence, the intron and various restriction endonuclease sites, and the amino acid sequence (SEQ ID No. 28) corresponding to the gene that encodes for the 38kD esterase isolated from Aspergill us ni ger.

Figure 6 illustrates the DNA sequence of the gene coding for the esterase 38kD (SEQ ID No. 29).

Figure 7 illustrates a southern spotting gel showing hybridization between a DNA probe derived from the 38kD esterase of the invention and various other filamentous fungi ("gel") 1" ) .

Figure 8 illustrates a southern spotting gel showing hybridization between a DNA probe derived from the 38kD esterase of the invention and various other filamentous fungi ("gel 2" ) .

DETAILED DESCRIPTION OF THE INVENTION "Esterase" or "sterolitic activity" means a protein or peptide that exhibits a sterolitic activity, for example, those enzymes that have catalytic activity as defined in the classification of enzymes EC 3.1.1. The sterolitic activity can be shown by the ability of an enzyme or peptide to cleave the ester bonds, for example, the feruloyl, coumaroyl or acetyl-xylan groups, from organic compounds in which they are known to exist, for example, primary and secondary cell walls. Preferably, the esterase comprises a sterolitic activity that breaks down the ether bond of the phenolic esters such as: [5-0 - ((E) ~ feruloyl) -aL-arabinofur nos i 1] (1-3) -O-β -D-xylopyranosyl- (1- > 4) -D-xylopyranose (also known as FAXX); [5-0- ((E) -feruloyl) -a-L-arabinofurans i1] (1-> 3) -O-β-D-xylopyranose (also known as FAX); O-β-xylopyranosyl- (1-4) -0- [5-0- ((E) -feruloyl) -a-arabinofurans i 1- (1 -3)] -0-β-D-xylopyranosyl) - (1-> 4) -D-xylopyranose (also known as FAXXX); [5-0- ((E) -p-coumaroyl) -a-L-arabinofuranosyl] (1-> 3) -O-β-D-xylopyranose (also known as PAX); O-ß-D-xylopyranosyl- (1- »4) -0- [5-O- ((E) -p-coumaroyl-α-arabinofuranosyl- (1-> 3)] -0-β-D- xylopyranosyl- (1-> 4) -D-xylopyranose (also known as PAXXX) and other fenslic acid oligosaccharides with ester linkage as are known in the art Such esterases are generally termed ferulic acid esterase (FAE) or enzymes having feruloyl esterase activity It has surprisingly been found that an esterase having ferulic acid esterase activity, which can be purified from Aspergillus niger, as described herein, and having a amino acid sequence as shown in figure 5, it also has activity on the sugar beet pulp and also proteolytic and lipolytic activity. Thus, according to a particularly preferred embodiment of the present invention, an esterase and / or a DNA encoding that esterase is provided, whose esterase also has lipolytic and / or proteolytic activity. Accordingly, the esterase of the invention having measurably sterolitic sterolitic activity on the feruloyl and coumaroyl esters, also has proteolytic and lipolytic activity. Preferably, the esterase and / or the DNA encoding the esterase according to the present invention is derived from a fungus, more preferably from an anaerobic fungus and more preferably from Aspergillus spp, for example, Aspergillus niger. Thus, it is contemplated that the esterase or DNA encoding the esterase according to the invention can be derived from Absidia spp: Acremonium spp; Actinomycetes spp: Agaricus spp; Anaeromyces spp; Aspergillus spp; including A. auculeautus, A. awamo, A. flavus, A. foetidus, A. fumaricus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreus and A. versicolor, Aeurobasidium spp; Caphalosporum spp; Chaetomium spp: Coprinus spp; Dactyllum spp; Fusarium spp; including F. conglomerans, F. decemcellulare, F. j avanicum, F. lini, F. oxysporum and F. solani; Gliocladium spp; Humicola spp, including H. insolens and H. lanuginosa; Mucor spp; Neurospora spp, including N. crassa and N. sitophila; Neocallimastix spp; Orpinomyces spp; Penicillium spp; Phanerochaete spp; Phlebia spp; Piromyces spp; Pseudomonas spp; Rhizopus spp; Schizophyllum spp; Streptomyces spp; Trametes spp; and Trichoderma spp; including T. reesmi, T. longibrachiatum and T. viride; Y Zygorh.yncb.us spp. Similarly, an esterase and / or the AD? which codes for an esterase as described herein, can be found in bacteria such as Streptomyces spp, including S. olivochromogenes; specifically ruminal bacteria that degrade fiber such as FijbroJbacter succinogenes; and in yeasts including Candida torresli, C. parapsllosis; C. sakm; C. zeylanoides; Pichia minuta; Rhodotorula glutinis; R. mucilaginosa; and Sporobolomyces holsaticus. According to the preferred embodiment of the invention, the esterase is in a purified form, for example, present in a particular composition in a concentration higher or lower than that which exists in an organism of natural or wild-type origin, or in combination with the components not normally present after expression from an organism of natural or wild-type origin. "Expression vector" means a DNA construct comprising a DNA sequence that is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence for controlling such transcription, a sequence encoding the appropriate ribosome binding sites on mRNA, and the sequences that control the termination of transcription and translation. Different cell types with different expression vectors are preferably used. A preferred promoter for the vectors used in Bacill us subtili s is the AprE promoter; a preferred promoter used in E. col i is the Lac promoter and a preferred promoter used in Aspergill us niger is gl aA. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into an appropriate host, the vector can replicate and function independently of the host genome, or it can, under suitable conditions, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used interchangeably. However, it is intended that the invention include other forms of expression vectors that serve equivalent functions and which are, or become known in the art. Thus, a wide variety of host / expression vector combinations can be employed in the expression of the DNA sequences of this invention. Useful expression vectors, for example, can consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences such as various known SV40 derivatives and known bacterial plasmids, for example, E plasmids. coli including El, pCRl, pBR322, pMb9, pUC 19 and its derivatives, plasmids of a wider range of hosts, for example, RP4, phage DNAs for example, numerous phage derivatives, eg, NM989, and other phage DNA, for example, M13 and single-stranded filamentous DNA phages, yeast plasmids such as 2μ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in animal cells, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ DNA phage or other expression control sequences. Expression techniques using the expression vectors of the present invention are known in the art and are generally described, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989) . Frequently, such expression vectors that include the DNA sequences of the invention are transformed into a unicellular host by direct insertion into the genome of a particular species through an integration event (see for example Bennett and Lasure, More Gene Manipulations in Fungi, Academic Press, San Diego, pp. 70-78 (1991) and articles cited therein which describe directed genomic insertion in fungal hosts, incorporated by reference herein). "Host strain" or "host cell" means a suitable host for an expression vector that comprises the DNA according to the present invention. Host cells useful in the present invention are generally prokaryotic or eukaryotic hosts, including any transformable microorganism in which expression can be achieved. Specifically, the host strains may be Bacillus subti ls, Escheri chi al coli, Tri chodmrma l ongibrachi a tum, Saccharomyces cerevi si ae or Aspergillus niger, and preferably Aspergi l lus ni gmr. The host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of replicating vectors coding for esterase and its variants (mutants) or expressing the desired peptide product. "Derivatives" means a protein that is derived from a precursor protein (e.g., the native protein) by adding one or more amino acids to either the C-terminus or the N-terminus and replacing one or more amino acids one s a number from different sites in the amino acid sequence, deletion of one or more amino acids at one or both ends of the protein, or at one or more sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of an enzymatic derivative is preferably carried out by modifying a DNA sequence encoding the native protein, transforming that DNA sequence into a suitable host, and expressing the modified DNA sequence for form the enzyme derivative. The derivative of the invention includes peptides comprising altered amino acid sequences in comparison with an amino acid sequence of enzymatic precursor (e.g., a wild-type or native-state enzyme), whose peptides retain an enzymatic nature characteristic of the precursor enzyme, but which have altered properties in some specific aspect. A "derivative" within the scope of this definition will generally retain the characteristic sterolic activity observed in the native or progenitor form, to the extent that the derivative is useful for similar purposes as the native or progenitor form. However, it is further contemplated that such derivatives may have altered substrate specificity, eg, higher or lower affinity for a specific substrate such as the feruloyl, cinnamoyl or coumaroyl groups, or modified pH, modified temperature or oxidative stability. The derivative of the invention can also be produced through chemical modification of the precursor enzyme to alter the properties of the same. Hybridization is used herein to analyze whether a given fragment or given gene corresponds to the esterase described herein, and thus falls within the scope of the present invention. The hybridization assay is essentially as follows: Genomic DNA from a particular target source is fragmented by digestion with one or more restriction enzymes, for example, EcoRI, HindIII, PinAI, Mlul, Spel, BglII, PpulOI, Mfel, Ncol, Blnl, Eagl and Xmal (supplied by New Enland Biolabs, Inc., Beverly, MA and Boehringer Manhei) according to the manufacturer's instructions. The samples are then subjected to electrophoresis through an agarose gel (such as, for example, 0.7% agarose) so that the separation of the DNA fragments can be visualized by size. The gel can be briefly rinsed in distilled water and subsequently depurinated in an appropriate solution for 30 minutes (such as, 0.25 M HCl) with gentle stirring followed by denaturation for 30 minutes (eg, in 0.25 M NaOH) with gentle agitation. The DNA can then be transferred onto a suitable positively charged membrane, for example, the Maximum St ength Nytran Pl us membrane (Schleicher and Schuell, Keene, NH), using a transfer solution (such as, for example, sodium hydroxide). 0.4 M). After the transfer is complete, generally after 2 hours or more the membrane is rinsed and air-dried at room temperature after using a rinse solution (such as, for example, 2X SSC [2X SSC = 300 NaCl mM, trisodium citrate 30 mM]). The membrane should then be prehybridized (for approximately 2 hours or more) in an appropriate prehybridization solution (such as, for example, in aqueous solution containing 100 ml; 20-50 ml formamide, 25 ml 20 X SSPE ( IX SSPE = 0.18 M NaCl, 1 mM EDTA, 10 mM NaH2P04, pH 7.7), 2.5 ml of 20% SDS, 1 ml of 10 mg / ml herring sperm DNA, 21.5 ml of distilled water). As would be known to one of skill in the art, the amount of formamide in the solution of Prehybridization must be varied depending on the nature of the reaction obtained according to routine methods. Thus, a smaller amount of formamide can result in a more complete gel in terms of identification of hybridization molecules than the same procedure using a larger amount of formamide. On the other hand, a strong hybridization band can be more easily identified visually by the use of more formamide. The DNA probe derived from the sequence of Figures 5 or 6 should be isolated by electrophoresis in 1% agarose, the fragment removed from the gel and recovered from the excised agarose. This purified fragment of DNA is then labeled with 32P, randomly primed (using, for example, the dialing system Magaprime according to the manufacturer's instructions (Amersham International foot, Buckinghamshire, England). The labeled probe is denatured by heating at 95 ° C for 5 minutes and immediately added to the previous prehybridization solution containing the membrane. The hybridization reaction must proceed for an appropriate time and under appropriate conditions, for example, for 18 years at 37 ° C with gentle shaking. The membrane is rinsed (for example, in 2X SSC / 0.3% SDS) and then washed with an appropriate washing solution and with gentle agitation. The desired requirement will be a reflection of the conditions under which the membrane (filter) is washed. Specifically, the requirement of a given reaction (eg, the degree of homology necessary for successful hybridization) will depend on the washing conditions to which the filter from Southern blotting is subjected after hybridization. The conditions of "Low demand" as defined in this, will comprise washing a filter from a Southern stain with a solution of 0.2X SSC / 0.1% SDS at 20 ° C for 15 minutes. The "Standard Requirement" conditions comprise an additional washing step comprising washing the filter from the Southern Stain, a second time with a 0.2 x SSC / 0.1% SDS solution at 37 ° C for 30 minutes. Figures 5 and 6 illustrate the amino acid sequence and DNA sequence of a novel esterase derived from Aspergillus niger. Isolated esterase has a molecular weight of approximately 38kD (as shown on SDS-PAGE), a pl of about 2.8 (as shown on IEF), an optimum pH of about 5.1 on methyl ferulate, an optimum temperature of about 55 ° C and activity on coumaroyl esters and feruloyl, and sugar beet pulp. The FAE gene shown in Figure 5 (SEQ ID No. 27) is approximately 2436 base pairs in length, including the inferred intron sequence and, if expressed, will encode the esterase encoded herein from Aspergi ll us ni ger (hereinafter the "38kD esterase"). For purposes of the present invention, the term "esterase 38kD" means an esterase derived from Aspergillus us niger corresponding to the esterase specifically exemplified herein. The DNA provided in Figures 5 or 6 will be useful for obtaining homologous DNA fragments from other species, and particularly from anaerobic fungi, which encodes an enzyme that has estergic activity. The DNA sequences of the present invention can be expressed by operatively linking them to an expression control sequence, in an appropriate expression vector, and employed in this expression vector to transform an appropriate microbial host according to well-established techniques in the art. The polypeptides produced on the expression of the DNA sequences of this invention can be isolated from the fermentation of the cultures of animal cells and purified in a variety of ways according to well-established techniques in the art. Someone skilled in the art is able to select the most appropriate isolation and purification techniques. The esterase isolated according to the present invention is useful in applications in which it is desired to remove the phenolic constituents of the xylan oligosaccharides. For example, esterases can be applied to improve animal nutrition, and possibly human nutrition, since the digestion capacity of forage cell walls appears to be dependent on the phenolic forage content. In addition, esterases could be applied in the pulp industry of paper as hydrolysis of the portions linked to the phenolic ester from lignin, which can contribute to the solubilization of lignin and they can also contribute to the hydrolysis of lignin / hemicellulose bonds. Esterases may be of potential use in the synthesis of carbohydrate derivatives and in the bioconversion of agricultural residues to fermentable sugars and free phenolic acid, useful as an antioxidant, photoresist and / or antimicrobial in food and personal care products; as a feedstock for the conversion to flavoring biopolymers (such as vanilla) and valuable chemicals. Esterases have also been implicated in the finishing of textile fibers (see for example, PCT Publication No. 96/16136). The activity of esterases towards multiple substrates, present in many powder-based strains, their activation by surfactants and their specificity towards phenolics, suggests that esterases can also be of value in detergents. The availability of relatively large amounts of esterase, facilitated by the present invention will enable the development of additional valuable applications. The invention will be explained later in the appended examples, which are provided for illustrative purposes and should not be considered as limiting the invention.

EXAMPLES EXAMPLE 1 Purification and Isolation of Pép -tidos that They comprised Acid Esterase Activity Ferulic and Design of DNA Fragments Degenerated for PCR A fermentation broth of Aspergi l lus niger was filtered (0.8 μm) and 10 ml was transferred to a centrifuge tube (50 ml) at room temperature. Saturated magnesium sulfate was added to give a final concentration at 60%. The solution was mixed and stored for approximately one hour at 4 ° C and then centrifuged at 1500 x g for 20 minutes at 4 ° C. The supernatant was removed and the button was resuspended in distilled water. Four tubes prepared as described above were combined and diluted to approximately 200 ml with ammonium sulfate (2 M) to give a final concentration of ammonium sulfate of 1.2 M, the pH was adjusted to pH 7.4 by the addition of Tris-? Cl (200 mM). The enzyme sample was subjected to chromatography by hydrophobic interaction chromatography (Poros® HPEM phenyl ether, Perseptive BioSystems, perfusion chromatography, 12 x 30 cm). The column was connected to a BioCad® Perfusion Chromatography Workstation (Perseptive BioSystems) and equilibrated with 5 volumes of Tris-HCl columns (50 mM, pH 7.4) plus 1.2 M ammonium sulfate. The sample (205 ml) was applied to the column and separated at a flow rate of 30 ml / min with a linear gradient from 1.2 M to 200 M ammonium sulfate on 20 column volumes. Fractions (15 ml) were collected during the gradient phase of the separation and tested for FAE activity with methyl ferulate by the method of Faulds and Willamson (1994, Microbiology 140: 779-787). Four fractions eluted at 750 mM of ammonium sulfate contained 83% of the initial AED activity. Active fractions were combined (60 ml), dialysed by ultrafiltration in initial buffer for the next chromatographic step (10 kDa membrane, 20 L sodium acetate buffer 25). mM pH 5.0). The sample was concentrated to 10 ml in the preparation for ion exchange chromatography. The ion exchange chromatography was performed using a strong MonoQ anion exchanger (MonoQ®, HR 10/10, Pharmacia Biotechnology) connected to a BioCAD Perfusion Chromatography Workstation and equilibrated with 25 mM sodium acetate (pH 5.0 ). The sample (10 ml) was applied to the column and eluted at a flow rate of 5 ml / min with a linear gradient of sodium chloride (0-500 M) in 15 column volumes. Fractions (5 ml) were cut during the gradient and tested for FAE activity (activity against feruloyl esters). The activity of FAE eluted as a single peak at 155 M sodium chloride was collected in a fraction. The sample was concentrated (Centricon, 10 kDa) up to 1.5 ml. High resolution size exclusion chromatography (HPSEC) was carried out using two Superdex 75 columns (10/30 HR, Pharmacia Biotechnology) connected in tandem on a BioCAD Perfusion Chromatography Workstation. The columns were balanced with 10 column volumes of sodium acetate buffer (25 mM, pH 5.0) containing 125 M sodium chloride and 0.01% triton X-100. Columns were calibrated for determination using protein standards of known molecular mass (Bio-RAD filtration standards, and Sigma gel filtration standards). Samples were applied (500 μl) and separated at a flow rate of 750 μl / min. Fractions (1 ml) were collected. The activity of FAE eluted with a single peak corresponded to a molecular mass of approximately 32 kDa. After the native PAGE of an active fraction, desalted, from HPSEC, a single protein band was observed. The isoelectric point of FAE was determined using Dry IEF in Phast gel balanced with an ampholyte in solution (final concentration of 20% containing a mixture of pl 2-4, 80%, pl 3-10 20%) Glycerol (10%) for 1 hour. The separation was carried out following the recommendations of the manufacturers (IEF Dry Instructions Bulletin of Pharmacia Biotechnology) and with Coomassie R-250. The sample migrated as a single protein band with an isoelectric point of about 2.8.

The Western spots of the FAE sample of HPSEC were made using PVDF membranes (0.2 μm pore size), a Novex mini-gel apparatus for obtaining the N-terminal amino acid sequence by methods recommended by the manufacturer. A sample of the AED was dialysed in buffer (5 mM MES, pH 5.8). The resulting purified protein was placed in an aqueous solution for analysis of the peptide sequence according to standard methods. Briefly, the peptides were digested in solution with the following sequencing-grade proteases: Lys-C-200 μl of reaction buffer comprising 100 mM ammonium bicarbonate and 2-4 μg of enzyme, pH 8.0, overnight at 37 ° C; Arg-C-200 μl of reaction buffer comprising Tris 20 M and 4 μg of enzyme, pH 7.5 + 1.5 mM calcium chloride + 2 M DTT overnight at 37 ° C; Glu-C-digestion buffer for digestions on spot was 50 mM ammonium bicarbonate with 4 μg enzyme, 10% acetonitrile and 1% reduced triton X-100.

Cleavage with CNBr-was conducted by dissolving the enzyme sample in 200 μl of 70% formic acid in water and CNBr crystals added in sufficient quantity to produce cleavage of the etisin. The digested peptides were then concentrated to approximately 100 μl and loaded directly onto a reverse phase HPLC. (column C18 Phenomenex Primersphere, 250 x 2.0 mm). Reverse phase separations were carried out using the Applied Biosystems 140A Solvent Distribution System. The buffers used were 0.1% trifluoroacetic acid (TFA) in water (A), 70% acetonitrile in water + 0.070% TFA (B), the flow rate was 150 μl per minute with a gradient as follows: 0 minutes -Buck absorber B al- 5%, 10 minutes - Shock absorber B, at 10%, 80 minutes - Shock absorber B, at 80%, 85 minutes - Shock absorber B, at 100%, 90 minutes - Buffer B at 100%. The digestions with CNBr are treated as follows: water is added to the solution and the total volume is concentrated to 10 μl in a rapid vacuum apparatus. Additional water is added to approximately 1 ml and it dries again until approximately 100 μl. This removes most of the formic acid / CNBr. The various peptide fragments obtained as described above were analyzed to determine their sequence and for the subsequent development of the degenerate probes for use in the cloning of the gene coding for the 38kD esterase from the genome of the donor organism. The peptide sequence analysis of 38kD esterase was problematic due to the cycles containing mixed signals, indicating the presence of multiple polypeptides in the sample analyzed. The sequencing of the protein resulted in an N-terminal sequence and several additional peptide fragments, as follows: ASTQGISEDLYSRLVEMATISQAAYXDLLNIP (SEQ ID No. 1) XTVGFGPY (SEQ ID No. 2) FGLHLXQXM (SEQ ID No. 3) XISEDLYS (SEQ ID No. 4) YIGWSFYNA (SEQ ID No. 5) GISEDLYXXQ (SEQ ID No. 6) XISESLYXXR (SEQ ID No. 7) GISEDLY (SEQ ID No. 8) LEPPYTG (SEQ ID No. 9) XANDGIPNLPPVEQ (SEQ ID No. 10) YPDYALYK (SEQ ID No. 11) From these fragments, degenerate probes suitable for hybridization and use as PCR primers were produced and fragments were obtained that were believed to be derived from the gene coding for esterase 38kD. However, the sequencing of the fragments obtained in this way (500 and 100 base pairs) showed that the fragments were merely PCR artifacts and were not useful in cloning the 38kD esterase. Further analysis of 2 different probes derived from 2 isolated protein sequences as described above, resulted in similar lack of success. From these results, it was determined that routine protein purification and peptide sequencing procedures were insufficient to obtain suitable peptide segments for the preparation of degenerate DNA probes.

The inventors herein hypothesized that a specific property of the protein or purified protein composition was the prevention of obtaining the purified representative protein. To test this theory, the aforementioned protein product was analyzed by means of isoelectric focusing gel at pH 2-4 under various conditions. As shown in Figure 3, the protein samples taken from the purification steps along the purification method described above appear to be a single band of highly purified protein. A second analysis was carried out in which the purified protein was subjected to denaturing conditions of an SDS-PAGE and the results of western blotting. As shown in Figure 1, the resulting protein showed a number of bands indicating either some degeneration of the protein or the other compounds hidden during the IEF gel. The sequencing of each of the numerous bands showed that each possessed an identical N-terminal sequence and that proteolysis appeared to be occurring from the carboxyl terminus.

From the data, the present inventors hypothesized that numerous fragments may be appearing due to the carboxyl-terminal proteolytic cleavage within the same molecule, after the unfolding of the protein in the reduced SDS buffer. The unfolding of the 38kD esterase can expose the previously internal hydrophobic residues, for example, tyrosine, tryptophan and nilalanine, providing substrates structurally similar to the ester-linked feruloyl group, which could be recognized at the active site of the 38kD esterase allowing hydrolysis of the peptide. This result was highly unpredictable due to the fact that the enzymatic action observed to date of the isolated protein was sterolitic and not proteolytic. In any case, the inventors herein launched the theory that if the conditions of protein denaturation (eg, unfolding of the peptide chain) are avoided, internal truncation can be avoided. To do this, the purified protein from the ion exchange chromatography step was subjected to additional chromatography using size exclusion chromatography. high resolution (HPSEC as described above). The 38kD esterase purified by HPSEC was separated by SDS-PAGE and a Western blot on a PVDF membrane that was performed for "on spot" sequencing. The digests directly from the spots were prepared as follows: pure trifluoroacetic acid was added to the spot containing the solution to give a final volume containing 50% TFA. This solution is then sonic for 5 minutes. The liquid (but not the staining parts) was removed, and a 50% acetonitrile solution in 0.1% TFA was added. The sample is sonicated again for 5 minutes. The liquid was removed and replaced by a final wash of 0.1% TFA in water and a final sonication of 5 minutes. All wash solutions were combined and concentrated to approximately 100 μl. This method allowed polypeptides resulting from enzymatic digestions to be collected without additional proteolysis by the 38kD esterase immobilized on the membrane. In this way, the simple polypeptides suitable for the sequential analysis were obtained due to the esterase of 38kD which is immobilized on the PVDF, thereby preventing carboxyl-terminal proteolytic cleavage or truncation and the presence of mixed amino acid signals during each sequencing cycle. When this procedure was followed, a number of fragments were produced that were appropriate for the design of the degenerate DNA fragments.

EXAMPLE 2 Isolation of a Fragment of 650 Pairs of Bases That Corresponds to the FAE Gene Based on the peptide fragments obtained in Example 1 after cleavage of the protein, the problem had been solved, the gene coding for the 38kD esterase was cloned by amplifying the gene from its genome using the reaction in polymerase chain and the degenerate oligonucleotide primers were designed appropriately. The primers were designed based on the partial amino acid sequences of the fragmented 38kD protein esterase. The amplification of three fragments from the 38kD esterase gene was obtained using the following four oligonucleotide primers. The oligonucleotide primers were designed based on the underlined peptide sequence after the oligonucleotide primers. The following abbreviations were used to identify the alternations in the equilibrium position: I = inosine, W = A / T, S = C / G, R = A / G, Y = T / C, H = A / T / C , D = A / G / T, X = A / T / G / C.

Primer 11 in CGGGAATTCGCIWSIACICARGGXAT sense: (SEQ ID No. 12) Derived from: ASTQGISEDLYSRLVEMATISQAAYA7DLLNIP (SEQ ID No. 13) Primer 7 in CGGGAATTCTAYTAYATHGGITGGGT sense: (SEQ ID No. 14) Derived from: VHGGYYIGWVSVQDQV (SEQ ID No. 15) Primer 8 antiCGGGAATTCACCCAICCDATRTARTA sense: (SEQ ID No. 16) Derived from: VHGGYYIGWVSVQDQV (SEQ ID No. 17) Primer 2 antiCGGGAATTCTTIGGIA ICCRTCRTT sense: (SEQ ID No. 18) Derivative of; TDAFQASSPDTTQYFRVTHANDGIPNL (SEQ ID No. 19) Two primers made it possible to deduct putative fragments of amplified DNA coding for the 38kD esterase: Primer 3 antiCGGGAATTCATICCRTCRTTIGCRTG sense: (SEQ ID No. 20) Derived from: TDAFQASSPDTTQYFRVTHANDGIPNL (SEQ ID No. 21) Primer 12 antiCGGGAATTCGCYTGRAAIGCRTCIGTCAT sense: (SEQ ID No. 22) Derived from: (M) DAFQASSPDTTQYFRVTHANDGIPNL (SEQ ID No. 23) An EcoRI restriction endonuclease recognition site and a "GC" clamp at the 5 'end of all the primers were included to facilitate cloning of the amplified fragments within the plasmid vector pUCld. The PCR reaction included placement in the following in "Hot Star" tubes (Molecular Bio-Products, Inc., San Diego, CA) in the order provided: 1 μl of primer in the 500 ng / μl direction 1 μl of anti-sense primer 500 ng / μl 2 μl of nucleotide mixture (1 OmM of each dNTP) 5 μl of PCR buffer lOx 41 μl of distilled water Warmed to 95 ° C by 90 seconds, placed on ice for 5 minutes. 5 μl of PCR buffer lOx 43 μl of distilled water 1 μl of genomic DNA of Aspergil l us niger 1 μl of DNA-polymerase Taq (Boeheringer Mannheim, 5 U / μl) The amplification was carried out in a PTC-150 Model Minicigrapher (MJ, Research Inc., Watertown, Mass.). The amplification conditions followed a sequential pattern of 95 ° C for five minutes; 40 ° C for 90 seconds; 72 ° C for 3 minutes; and 28 cycles of 94 ° C for 1 minute, 40 ° C for 90 seconds, and 72 ° C for 3 minutes for 28 cycles. HE included a final extension step of 72 ° C for _ 2 minutes. The primers were used for PCR amplification in the following pairing combinations: 11-2, 11-3, 8, 11-12, 7-2, 7-3 and 7-12. Each primer combination produced multiple bands of DNA after electrophoresis on agarose. Larger DNA bands for the PCR products of primer pairs 7-2, 3-3 and 7-12 were present around 350, 350 and 300 base pairs respectively, as visualized on a. 3% NuSieve agarose electrophoresis gel (FMC Corp.). Anti-sense primers 2, 3 and 12 were designed for the same continuous peptide fragment: (M) TDAFQASSPDTTQYFRVTHANDGIPNL (SE ID No. 24). The anti-sense primers 2 and 3 encode almost for the same stretch of DNA, their 3 'ends are displaced only by 6 bases. An anti-sense primer 12 corresponds to the amino acids that are upstream of the primers 2 and 3, the 3 'end of the anti-sense primer 12 is displaced by approximately 60 base pairs of the primers 2 and 3. Therefore, the lengths of the PCR band were approximately consistent with a stretch continuous DNA coding for 38kD esterase. In addition, pairs of primers 11-2, 11-3 and 11-12 produced bands of approximately 650, 650 and 600 base pairs respectively. These lengths were approximately consistent with the amplification of a piece of DNA encoding the 38kD esterase. The PCR amplification products were digested with EcoRI, ligated into the pUCld cloning vector and then transformed into E. col i. The cloned PCR products were sequenced. The sequencing of the product of the primers 11-2 revealed a DNA sequence of 650 base pairs shown in Figure 2 (SEQ ID No. 25) which after translation codes for 197 amino acids. A total of 155 residues corresponded to nine sequenced peptide fragments of the 38kD protein esterase. There is a putative 57-base pair intron that contains splicing sequences of GTATGC at the 5 'site, an internal lacing sequence of CACTAACT, and TAG at the 3' splice site. In addition, a product of primers 11-8 when sequenced reveals approximately the same 314 bases (5'-3 ') of fragment 11-2 of 650 pairs of bases. A product of 350 base pairs of primers 7-2 revealed the DNA corresponding in sequence to the second half of the 11 -2 fragment of 650 base pairs.

EXAMPLE 3 Obtaining Genomic DNA from Aspergill us ni ger for Cloning A preserved culture of Aspergillus niger was developed on Papa Dextrose Agar (PDA) at 30 ° C. Approximately 2 cm 2 of the fungi developed on PDA were inoculated in 50 ml of Yeast Extract Glucose medium in a 250 ml protected flask, and incubated at 33 ° C on a rotary shaker at a speed of 300 RPM for 24 hours. The mycelia were harvested through a miracloth cloth, squeezed, immediately frozen in liquid nitrogen and crushed with half a teaspoon of sand in a mortar and pestle for approximately 2 minutes. Genomic DNA was extracted from the crushed mycelium using a modification of the Genomic Easy DNA Isolation Kit from Invitrogen. Mycelium frozen and prepared was immediately transferred to a centrifuge tube to which 3.5 ml of a solution A was added, followed by swirling and a 10 min incubation at 65 ° C. Next, 1.5 ml of Solution B was added, followed by swirling. 5 ml of chloroform was added, followed by swirling until the viscosity decreased and the mixture was homogeneous. The mixture was centrifuged at 15,000 x G at 4 ° C for 20 minutes. The upper phase was transferred to a new tube and precipitated with two volumes of 95% ethanol. The precipitation reaction was incubated on ice for 30 minutes. The precipitation DNA was concentrated by centrifugation at 15,000 X G at 4 ° C for 15 minutes. The ethanol was removed. The DNA button was washed with 25 ml of 70% ethanol and the mixture was centrifuged at 15,000 X G at 4 ° C for 5 minutes. The 70% ethanol was removed and the pellet or button was allowed to air dry for 5 minutes. The extracted DNA was suspended in a volume of 500 μl of TE, RNAse was added to a final concentration of 4 μg / ml. This extracted genomic DNA was used in PCR amplification of the DNA fragments encoding the 38kD esterase.

EXAMPLE 4 Use of the Fragment Obtained from 650 Pairs of Bases for Ai if the DNA Coding for Enzymes Homoloids from Aspe • rgill U S or Others Species A particularly effective method for obtaining clones of homologous genomic DNA is by the construction and selection of a subgenomic library. In summary and as described in more detail, below, this method involves cutting the genomic DNA to completion with the appropriate restriction endonuclease, performing Southern hybridization with the 650 base pair fragment with a probe, binding the fragments of appropriate size within a plasmid vector, transforming the plasmid into E. col i and then probed with Southern colonies with the 650 base pair fragment to obtain a genomic clone. These techniques are known in the art and are described in Current Protocols in Molecular Biology, supra. To obtain clones of a vector comprising the gene coding for the complete 38 kD protein esterase, genomic DNA was prepared of Aspergill us ni ger as in example 3. The genomic DNA was fragmented by digestion with a number of restriction enzymes: EcoRI, HindIII, PinAI, Mul, Spel, BglII, PpulOl, Mfel, Ncol, BInl, Eagl and Xmal ( supplied by New England Biolabs, Inc., Beverly, MA and Boehringer Manheim). The reaction conditions combined 3 μl of genomic DNA, 2 μl of the appropriate restriction endonuclease buffer 10X (according to manufacturers' instructions), 2 μl of the restriction enzyme (at 10 units / μl), 13 μl of distilled water, the reaction proceeded at 37 ° C for 6 hours. The samples were then subjected to electrophoresis through a 0.7% agarose gel, so that the separation of the DNA fragments can be visualized between a size of 1 kb up to plus 12 kb. The gel was briefly rinsed in distilled water and subsequently depurinated for 30 minutes in a 0.25 M HCl solution with gentle agitation, followed by denaturation for 30 minutes in a 0.4 M sodium hydroxide solution, with gentle agitation. The DNA was then transferred onto a positively charged High Strenght Nytran Pl membrane (Schleicher &; Schuell, Keene, N.H) using a solution of 0.4 M sodium hydroxide as a transfer solution. After the transfer was completed, more than two hours, the membrane was rinsed in 2X SSC and air dried. The membrane was then prehybridized for 8 hours in a prehybridization solution containing 100 ml: 50 ml of formamide, 25 ml of 20X SSPE (IX SSPE = 0.18 M NaCl, 1 mM EDTA, 10 mM NaH2P0, pH 7.7), 2.5 my SDS at 20%, 1 ml of 10 mg / ml, herring sperm DNA, cut, and 21.5 ml of distilled water. The cloned 650 base pair fragment of Example 2 was used as a membrane hybridization probe. The fragment was isolated from the pUCld plasmid by restriction digestion with EcoRI, electrophoresis in 1% agarose, excision of the gel fragment and recovery of the fragment from the excised agarose. This purified fragment of 650 base pairs of DNA was labeled with 32 P, with random priming using the Megaprime labeling system according to the manufacturer's instructions (Amersham International foot, Buckinghamshire, England). The labeled probe was denatured by heating at 100 ° C for 5 minutes, and immediately added to the solution of prehybridization that contained the membrane. The hybridization reaction proceeded for 18 hours at 37 ° C with gentle agitation. The membrane was rinsed in a 2X SSC / 0.3% SDS solution and then washed for 15 minutes in the same solution at 37 ° C with shaking. The membrane was further washed with a 0.2X SSC / 0.1% SDS solution at 37 ° C for 30 minutes. The membrane was then exposed to an X-Omat AR film (Eastman Kodak Co, Rochester, N.Y.) for 3 hours and was revealed. The revealed film of the digests prepared as described above, showed only one band of hybridization by restriction enzyme digestion, consistent with hybridization. Digestion with EcoRI showed a simple band of hybridization approximately at a length of 5.5 kb. Because this hybridized fragment was an excellent candidate fragment to contain a gene corresponding to the complete 38 kD esterase, as a result of its size that is consistent with a gene encoding a protein of that size, a subgenote library was constructed by choosing EcoRI for digest genomic DNA from Aspergi ll us ni ger, to obtain fragment sizes around 5.5 kb in length.

Restriction digestion was performed on the genomic DNA prepared as in Example 3. The reaction included 50 μl of genomic DNA, 50 μl of the 10X restriction endonuclease H buffer (Boehringer Mannheim), 25 μl of EcoRI (10 units / μl of Boehringer Mannheim), 375 μl of distilled water. The reaction proceeded at 37 ° C for 6 hours. The digestion mixture was subjected to electrophoresis through 0.8% agarose. Fragments between a range of approximately 5 kb and 6 kb were cut from the gel in three approximately equal slices. The three pools of the DNA fragments contained within the three gel slices each had a slightly different range of fragment lengths. DNA was recovered from the agarose slices using QIAquick gel extraction columns following the manufacturer's instructions (Qiagen, Inc., Chatsworth, CA). Approximately 1/10 of each pool of the recovered DNA was subjected to 0.8% agarose electrophoresis and Southern hybridized to the 650 base pair fragment, as described above. The DNA pool that gave the strongest hybridization signal was ligated into an EcoRI vector digested with E. coli (for example pLITMUS 28, New England Biolabs), which was then transformed into E. coli The transformants of E. coli were plated out on 5 plates of a concentration of approximately 500 colonies per plate (diameter of the plate 150 mm). The colonial surveys were carried out on the plates using the Maximum Strength Nytran Plus membranes. A Hybridization of Sourthern was performed using the 650 base pair fragment. Four strong hybridization signals were obtained. The colonies corresponding putatively to the four strong hybridization signals, were developed and their plasmid DNA was recovered. Restriction digestions on the plasmid DNA were performed using restriction enzymes that were chosen based on the sites within the 650 base pair fragment. A restriction digestion with plasmid gave restriction fragments consistent with the known restriction sites within the 650 base pair fragment. After DNA sequencing it was revealed that this clone contains the sequence of 650 base pairs that was obtained through the PCR described in Example 2. The mapping of restriction of this clone reveals the 650 base pair fragment that lies within approximately 5.5 kb of the cloned genomic DNA sequence. Based on this procedure, the DNA encoding the complete 38 kD esterase gene was isolated, corresponding to sequence provided in Figure 6 (SEQ ID No. 27) encoding a protein having the amino acid sequence of Figure 6 (SEQ ID No. 28). Modifications of this method that are known to produce similar results could also be effective for obtaining the appropriate DNA or clones. Of course, this method is similarly appropriate for the identification and cloning of homologous esterase enzymes from different species of Aspergi l l us ni ni ger. For example, as described above, a genomic library could be produced from a suitable microorganism by preparing the genomic DNA and cutting with an appropriate restriction endonuclease. The library or library could then be subjected to hybridization by Southern blotting with the 6.50 base pair fragment described in Example 2, as a probe, and the appropriate hybridization fragments ligated therein. of a limited expression vector, and transformed into a suitable organism, for expression. Suitable techniques for such processes are described, for example, in European Patent No. 215 594 (Genencor). EXAMPLE 5 Construction of an Expression System for FAE FAE production was achieved by constructing an expression method and transforming that vector into Aspergillus. The transformed strain of Aspergillus is then developed in an appropriate fermentation medium. An FAE expression vector is described below. The transformation of Aspergi l l us is known in the art and is described for example in "Cloning mapping and molecular analysis of the pyrG (orotidine-5 '-phosphate decarboxylase) gene of Aspergill us Ni dul ans ", B. Oakley et al., Gene, 61 (1987) pp. 385-399.A FAE expression vector can be constructed in E. coli plasmids available as pNEB193 (New England Biolabs, Beverly, MA These elements are required for the expression vector In summary, these elements are: The FAE gene with its downstream terminator sequence, a glucoalase promoter from A. niger and the pyrG gene of A. indulans, which is used as a selectable marker for transformation. The pyrG gene can be amplified by PCR from Aspergi l lus Ni dul FGSC4 obtainable from Fungi Genetics Reserve Center, Department of Microbiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7420 USA. The sequence of the FAE gene is given in Figure 6. The glucoamylase promoter of Aspergill us niger and the pyrG DNA sequences of A. nor dul ans can be obtained from the GenBank sequence database. The pyrG sequence of A. nidulans is described in Oakley et al. The DNA sequence of the glucoamylase promoter of A. niger is described in "Regulation of the GlaA gene of Aspergillus niger", Fowler et al., Current Genetics (1990) 18: 537-545. The elements are accommodated in the E plasmid. coli, in such a way that the glucoamylase promoter drives the expression of the fael gene, starting from the initial methionine codon fael (from the base 519 in the fael gene). This allows the glucoamylase promoter strongly promotes the expression of the FAE gene product. Methods for the construction of DNA sequences in E plasmids. col i, are known in the art. An acceptable method for the construction of an FAE expression vector in the vector pNEB193 is as follows: (a) PCR is used to amplify the pyrG gene of A. nor sweet ans and insert this sequence into pNEB193. This could be achieved with two primers and the appropriate conditions to obtain a pyrG fragment of approximately 2.0 kb in size. For example, the upper primer can be: '= GGCCTGCAGCCCCGCAAACTACGGGTACGTCC-3' (SEQ ID, No. 30) and the lower primer can be: 'CGCGCTGCAGGCTCTTTCTGGTAATACTATGCTGG-3' (SEQ ID No. 31) The genomic DNA of Aspergill us Ni dulans can be prepared for amplification as described above. The conditions necessary to amplify a 2.0 kb fragment are known in the art, for example these are given in "Expansion Fidelity PCR System" (Boehringer Mannheim, Indianapolis, IN). After amplification of the fragment, it is isolated and then digested with the PstI enzyme. Also plasmid pNEB 193 is digested with PstI. After digestion, the fragment and the plasmid are isolated and ligated together. (b) PCR is used to amplify the glucoamylase promoter of A. niger and place this sequence inside the constructed plasmid. This could be achieved by using two primers and the conditions to obtain a promoter fragment of approximately 1.9 kb in size. As examples of suitable primer, the upper primer could be '-GGCTTAATTAACGTGCTGGTCTCGGATCTTTGGCGG-3 '(SEQ.

ID. No. 32) and the lower primer could be '-GGGGCGCGCCAGATCTAGTACCGATGTTGAGGATGAAGCTC-3 '(SEQ ID No. 33). While many different strains are suitable for amplification, a strain particularly useful for amplification is strain ASCC 10864 from Aspergi llus niger (North American Collection of Species Crops (American Type Culture Collection, Rockville, Maryland).

A. ni ^ p r-a genomic amplification pu-ed-e-be isolated as described above. After amplification the fragment is isolated and digested with the enzymes PacII and AscI. The plasmid created in part (a) above is also digested with the enzymes PacII and AscI. The amplified fragment digestions of the plasmid could be ligated together. (c) Two fragments of the FAE gene are combined within the plasmid created in (b) using the EcoRI fragment of 5.5 kg comprising the complete FAE gene. The first fragment is created via PCR using the following primers in conjunction with the EcoRI 5.5 kb fragment of the FAE gene described above as the source to be amplified: forward primer 5 '-GCCCAGATCTCCGCAATGAAGCAATTCTCCGCCAAACAC-3' (SEQ ID No. 34) ) reverse primer: 5 '-AATAGTCGACGGAATGTTGCACAGG-3' (SEQ ID NO: 35) This fragment is digested with BglII and SalI to give or result a fragment of approximately 169 base pairs in length. The second fragment is prepared by incubation of the 5.5 kb EcoRI fragment of the FAE gene with Sali and EcoRI, the resulting 1.75 kb fragment being isolated. The plasmid created in (b) above is prepared for the insertion of the FAE gene by digestion with BglII and EcoRI. The three fragments of the 169 base pair PCR product, the 1.75 kb fragment and the stepped 2 plasmid digested with BglII / EcoRI are ligated together. This resulting plasmid could be an expression vector of the FAE gene of Aspergill us. The vector created above could be used to carry out the transformation of Aspergill us.

EXAMPLE 6 Identification of Homologous Genes in Fungi Filamentous A Southern hybridization experiment was performed under hybridization conditions described using 25% formamide in hybridization buffer as described herein. The FAE gene of 650 databases isolated from Example 2 was used to probe the digested genomic DNA from a genus number. The hybridization bands were obtained with the genomic DNA obtained from fungi different from Aspergillus niger, implying the existence of homologous esterase genes in these other organisms. Based on the hybridization data, it is believed that the DNA identified in this experiment will code for enzymes expressly related to sterolitic activity. The genes for these other homologous enzymes are cloned by the methods described. These cloned genes are then expressed in suitable hosts to introduce the encoded enzyme. Genomic DNA was digested with two restriction enzymes BglII and PpulOI, and then subjected to electrophoresis through 0.7% agarose in two different gels. The fragment sizes of the genomic DNA separated on the agarose gel were in the range of approximately 20 kb. The gels were depurinated and denatured, and subjected to Southern staining on Nytran plus. The membranes were air dried and hybridized with the 650P base pair fragment labeled with 32P. The membranes were washed under conditions of low demand, followed by washing under conditions of standard requirement. The membranes were then subjected to autoradiography. The reproduced gels are provided in Figures 7 and 8. gel 1 Band # Endonuclease DNA source 2 Digestion with BglII Aspergillus niger GCI strain # 7 3 Digestion with PpulOI Aspergillus niger GCI strain # 7 4 digestion with BglII Aspergillus terrus 5 digestion with PpulOI Aspergillus terrus 6 digestion with BglII Trichoderma reesei strain QM6a, ATCC13631 digestion with PpulOI Trichoderma reesei strain QM6a, ATCC13631 digestion with BglII Acremoni um ba chypeni um, ATCC32206 digestion with PpulOI Acremoni um bachypeni um, ATCC32206 17 digestion with BglII Aspergillus niger GCI strain # 7 18 digestion with PpulOI Aspergillus niger GCI strain # 7 19 digestion with BglII Gliocladium roseum 20 digestion with PpulOI Gliocladium roseum 25 digestion with BglII Pencillium notatum 26 digestion with PpulOI Pencillium notatum The bands are apparent in lanes 2, 3, 17 and 18 corresponding to the cloned FAE gene described in this patent. The bands appear in lanes 2 and 17 of digestion with BglII, which may indicate other homologous FAE enzymes present in the strain of Aspergill us niger. Two bands are present in lanes 4 and 5 and two bands are present in lane 4, indicating homologous DNA in Aspergillus terrus. One band is apparent in lane 7 indicating homologous DNA in Tri choderma reesel. One band is apparent in lane 8 indicating homologous DNA in Acremoni um brachypeni um. One band is apparent in lane 19 indicating homologous DNA in Gl i ocl adium roseum. Two bands are present in lanes 25 and 26 indicating homologous DNA in Peni cilli or tum note. EXAMPLE 7 Biochemical Properties and Specificity of Substrate of Purified AED according to Example 1 The 38kD esterase isolated according to Example 1 was analyzed for the biochemical properties. It was found that the molecular weight was of approximately 33kD as measured on SDS-PAGE and 30-32 kD as measured by HPSEL. The pl as measured by the isoelectric focusing gel (IEF) was found to be approximately 2.3. The purified 38kD esterase was found to be active towards various natural furoloyl and p-cumaroyl esters, the cell walls of wheat bran and the sugar beet pulp, wheat flour, the pentosan fraction of the wheat flour, and the ethyl and methyl esters of ferulic acid and p-coumaric acid. The kinetic data for the various substrates are presented in Table 1. The esterase of 3dkD showed an optimum pH of 5.1 for the methyl ferulate with d3% and 25% of maximum activity found at pH 3 and 8, respectively. When the 38kD esterase was incubated in a buffer for 30 minutes without substrate at pH 5.1, the optimum temperature was 55 ° C. With the 250 μM methyl ferulate present, the optimum increases to 65 ° C. A low Km of the FAXX trisaccharide favors the use of the 38 kD esterase of the present invention, in combination with a xylanase which leaves such carbohydrate oligomers preferably not hydrolyzed when the cell walls are degraded.

Purified 3dkd esterase was analyzed for a variety of biochemical activities with an API-20 enzyme test strip (BioMerieux Vitek) according to the manufacturer's instructions. The results are shown in Table 2. The activity was observed on the following substrates. "+++" indicates very strong response, "++" indicates strong response and "+ •" indicates activity shown towards the substrate, "-" means no activity detected.

TABLE 1 Activity of the 38kD Esterase on Diverso; Ferulolated oligosaccharides TABLE 2 Substrate Specificity for 36kD Esterase TABLE 2 Substrate Specificity for 38kD Esterase EXAMPLE 8 Activity of the 38kD Esterase to the Sugar Beet Pulp Substrate The pulp of sugar beet (100 mg of SBP) was incubated with FAE (1.5 FAXX Units as measured by the method described by McCallum et al., Analyti cal Bi ochemi s try, vol 196, p.362 (1991)) alone and in combination with 50 xylanase units from from Tri choderma l ongibrachi to tum (Irgazyme 4X, commercially available from Genencor International, Inc.) in sodium acetate buffer (100 mM, pH 5.0). The reaction mixtures were continuously inverted at 2 ° C during the incubation. The SBP was incubated with (i) buffer alone, (ii) xylanase alone, or (iii) boiled FAE served as control. The reactions were stopped at 12 and 24 hours by the addition of 1.1 equivalents of HCl. The determination of the total ferulic acid content of SBP was determined by saponification with sodium hydroxide by the method of Bormeman et al., App. My crobi ol. Bi or tech vol. 33, pp. 345-351 (1990). The ferulic acid released by the enzymatic treatment was determined by HPLC using standards Authors of ferulic acid (Aldrich) by the method of Bormeman et al., Anal. Bi ochem. vol. 190, pp. 129-133 (1990). The results are shown in table 3.

TABLE 3 Ferulic Acid Release from the Sugar Beet Pulp with Ferulic Acid Esterase 66 Of course, it should be understood that a wide range of changes and modifications can be made to the preferred embodiments described above. It is therefore intended that the above detailed description be understood in the context of the following claims, including all equivalents, which are intended to define the scope of this invention.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (32)

1. A DNA characterized in that it encodes a protein that has sterolitic activity that breaks the ester bonds of the phenolic esters.

2. The DNA according to claim 1, characterized in that the DNA is derived from a fungus, yeast or bacterium.

3. The DNA according to claim 2, characterized in that the DNA is derived from a fungus.

4. The DNA according to claim 3, characterized in that the DNA is derived from a filamentous fungus.

5. The DNA according to claim 4, characterized in that the DNA is derived from Aspergillus and comprises the feruloyl esterase.

6. The DNA according to claim 5, characterized in that the DNA comprises a partial sequence according to SEQ ID. No. 29

7. The DNA according to claim 1, characterized in that the DNA or part of the DNA encoding the amino acid sequence according to SEQ ID No. 28.

8. The DNA according to claim 1, characterized in that the DNA or part of the DNA encoding the amino acid sequence that is derived according to SEQ ID No. 28.

9. The DNA according to claim 1, characterized in that the DNA comprises at least one part that is capable of hybridizing under conditions of low demand with a DNA comprising all or part of the DNA sequence according to SEQ. ID. No. 29

10. The DNA according to claim 9, characterized in that the DNA is able to hybridize under conditions of standard requirement.

11. A method for the isolation of a DNA encoding a protein having esterase activity, characterized in that it comprises: (a) the creation of a library comprising fragments of a first DNA derived from a plant, animal, fungus, yeast or bacteria; (b) combining said library of the first DNA with a probe comprising a second DNA under conditions of low requirement to effect hybridization between the fragments in the DNA library and the probe, wherein the probe comprises the DNA corresponding to the I KNOW THAT. ID No. 29 or a portion thereof comprising at least 100 nucleotides.

12. The method according to claim 11, characterized in that the first DNA is derived from a filamentous fungus.

13. The method according to claim 11, characterized in that the first DNA is derived from Aspergi l l us.

14. The method according to claim 11, characterized in that the conditions suitable for hybridization comprise conditions of standard requirement.

15. The method according to claim 11, characterized in that the probe comprises DNA corresponding to a portion of SEQ ID No. 29 comprising at least 400 nucleotides.

16. DNA, characterized in that it is isolated according to the method according to claim 11.

17. DNA, characterized in that it is isolated according to the method according to claim 13.

18. DNA, characterized in that it is isolated according to the method according to claim 14.

19. The DNA, characterized in that it is isolated according to the method according to claim 15.

20. An expression vector, characterized in that it comprises the DNA according to any of claims 1 to 10.

21. A host cell, characterized in that it is transformed with the DNA according to any of claims 1 to 10.

22. A host cell, characterized in that it is transformed with the expression vector according to claim 22.

23. A purified esterase, characterized in that it is produced by the host cell according to claim 18 or 19.

24. A method for the production of an esterase, characterized in that it comprises the steps of: (a) transforming a suitable microbial cell with an expression vector comprising a DNA according to any of claims 1 to 10; (b) cultivating said transformed host cell under conditions suitable for the host cell to produce the esterase; (c) and, optionally, the separation of the esterase produced from the host cells to obtain a purified esterase.

25. A food supplement, characterized in that it comprises the enzyme produced by the method according to claim 24.

26. A process for the treatment of fabric, yarn or textile materials, characterized in that the fabric, the yarn or textile material is brought into contact with the enzyme produced according to claim 24.

27. A purified esterase, characterized in that it comprises the amino acid sequence provided in Figure 2 or a derivative thereof.

28. The purified esterase according to claim 27, characterized in that the esterase is from a filamentous fungus, bacterium or yeast.

29. The purified esterase according to claim 27, characterized in that the esterase is derived from Aspergill us.

30. The purified esterase according to claim 29, characterized in that the esterase is derived from Aspergill us niger.

31. The purified esterase according to claim 27, characterized in that the esterase corresponds to an esterase of 38kD.

32. A purified esterase, characterized in that it is encoded by the DNA according to any of claims 1 to 10.