Phenolic acid esterases, coding sequences and methods

This application is a Continuation-in-part of U.S. patent application No. 09/390,234, filed Sep. 3, 1999, now U.S. Pat. No. 6,365,390 which application claims priority from U.S. Provisional Application No. 60/099,136, filed Sep. 4, 1998.

This invention was made, at least in part, with funding from the United States Department of Energy (Grant No. DE-FG05 93ER 20127). Accordingly, the United States Government has certain rights in this invention.

The field of the present invention is the area of enzymes which degrade plant cell walls, and certain other substrates, in particular, the phenolic acid esterases, feruloyl esterases and/or coumaroyl esterase, and methods for using them in food compositions, feed compositions and supplements, nutriceuticals and in pulping.

Plant cell wall material is one of the largest sources of renewable energy on earth. Plant cell walls are composed mainly of cellulose, hemicelluloses, lignin and pectin. Arabinoxylan is one of the main constituents of hemicelluloses. It is composed of a chain of β(1→4) linked xylose units that are substituted by arabinose, acetate, and glucuronic acid. The arabinose has ester linked ferulic and p-coumaric acids [Bomeman et al. (1993) In: Hemicellulose and Hemicellulases, Coughlan and Hazlewood, Eds., pp. 85-102]. Ferulic acid has been shown to link hemicellulose and lignin [Ralph et al. (1995) Carbohydrate Research 275:167-178]. Feruloyl esterases are involved in breaking the bond between the arabinose and ferulic acid, thus releasing the covalently bound lignin from hemicelluloses. Feruloyl esterases have been found in many bacteria as well as fungi, but have not been extensively studied nor is there much sequence data available [Christov and Prior (1993) Enzyme. Microb. Technol . 15(6):460-75].

Clostridium thermocellum is a gram-positive bacterium that produces a multienzymatic structure termed the cellulosome. The cellulosome is one of the most active cellulose degrading complexes described to date. The cellulosome has a multi-polypeptide structure, including a scaffolding subunit which has nine cohesins binding to nine catalytic subunits, a dockerin domain for attachment to the cell wall, and a cellulose binding domain [Felix and Ljungdahl (1993) Annu. Rev. Microbiol . 47:791-819]. The catalytic subunits include endoglucanase, cellobiohydrolase, lichenase, and xylanase, many of which have been cloned and sequenced. They all have multidomain structures that include at least a dockerin domain for binding to the scaffolding domain, a linker, and a catalytic domain. They may also contain cellulose binding domains and fibronectin-like domains. There are reports that some enzymatic components may have more than one catalytic domain. Two of these are xylanase Y [XynY, Fontes et al. (1995) Biochem. J . 307:151-158] and xylanase Z [XynZ, Grépinet et al. (1988) J. Bacteriol . 170(10):4582-8]. XynY has a C-terminal domain whereas XynZ N-terminal domain without any functions determined. Although enzymes with dual catalytic domains (xylanase and βglucanase) have been found in other bacteria [Flint et al. (1993) J. Bacteriol . 175:2943-2951] neither phenolic acid esterase nor bifunctional enzymes have been found in C. thermocellum.

There is a need in the art for phenolic acid esterases, feruloyl esterases and/or coumaroyl esterases in pure form which degrade plant cell wall materials, and certain other substrates, for DNA encoding these enzymes to enable methods of producing ferulic acid and/or coumaric acid, as well as facilitating degradation of plant cell wall materials in the context of human, animal, fish or shellfish food, the pulping industry and in the area of nutriceuticals.

The present invention provides methods for improving nutrient availability in foods, especially plant-derived feedstuffs and foodstuffs with a significant non-starch polysaccharide content and/or with poorly digestible fiber. The methods comprise the step of combining the foodstuff or feedstuff with a feruloyl esterase as provided herein, desirably together with a xylanase, for example, the xylanase (XynA) protein derived from Orpinomyces PC-2. These enzymes can be prepared from their natural sources or the recombinant enzymes can be prepared using the teachings provided herein and in United States Patent No. 5,824,533 (Li et al., 1998) for the xylanase A of Orpinomyces PC-2. A foodstuff or feedstuff is combined with feruloyl esterase at a ratio of 0.1 to 200 units per kg dry weight of foodstuff or feedstuff. Where the xylanase A protein is also added, the xylanase ratio is 100 to 25,000 units per kg. An advantageous range is 500 to 10,000.U/kg or 1000 to 5000 U/kg. Where a beverage or liquid food or feed formulation is treated with feruloyl esterase or feruloyl esterase and xylanase A, the ratios are the same, with the calculation based on the dry weight of solids in the beverage or other liquid composition for consumption by a human or an animal.

The present invention further provides methods for improved pulping of plant material or recycled fiber materials, wherein the improvement comprises the step of adding a feruloyl acid esterase to the pulping mixture and incubating under conditions allowing enzymatic action of the ésterase on the non-starch polysaccharides in the mixture. The mixtures desirably contain a combination of feruloyl esterase and xylanase A. In pulping, the esterase is present in the pulping mixture at a ratio of 0.1 to 200 U/kg dry weight of pulp or fiber, and where xylanase A is present, the xylanase is present at a ratio of from 100 to 25,000 U/kg, desirably from 500 to 10,000, or from 1000 to about 5000 U/kg fiber or pulp dry weight.

The present invention further provides compositions comprising a feruloyl esterase protein. Desirably the composition further comprises a xylanase optionally xylanase A from Orpinomyces PC-2. These compositions can be liquid (non-concentrated solution or suspension) or dry (e.g., freeze-dried). The user then adds the liquid composition to food, feed or fiber, or the dry composition can be reconstituted before or upon addition to a food, feed or pulp.

The present invention further provides methods for improving the availability of ferulic acid from plant material for human, animal, fish or shellfish nutrition or health benefit. A feruloyl esterase composition described herein or a combination of a feruloyl esterase together with xylanase A is (are) added to a plant-derived foodstuff or feedstuff prior to consumption. Alternatively, a composition containing a feruloyl esterase digest or a feruloyl esterase—xylanase A digest of plant cell wall-containing material can be provided for use as a nutritional or nutriceutical supplement.

Herein are described novel phenolic acid esterases, having feruloyl esterase and coumaroyl esterase activities, and coding sequences for same.

One phenolic acid esterase corresponds to a domain of previously unknown function from xylanase Y of Clostridium thermocellum . The recombinantly expressed domain polypeptide is active and has an amino acid sequence as given in FIG. 1 as “XynY_Clotm.” The nucleotide sequence encoding the esterase polypeptide is given in Table 5, nucleotides 2383-3219, exclusive of translation start and stop signals. See also SEQ ID NOs:11 and 12.

A second phenolic acid esterase corresponds to a domain of previously unknown function of xylanase Z from C. thermocellum . The amino acid sequence of the esterase domain, which also is active when expressed as a recombinant polypeptide, is given in FIG. 1 as “XynZ_Clotm.” The nucleotide sequence encoding this polypeptide is given in Table 6, nucleotides 58-858. Further described is a phenolic acid esterase polypeptide additionally comprising a cellulose binding domain. A specifically identified cellulose binding domain has an amino acid sequence as given in Table 6, 289-400, with a corresponding coding sequence as given in Table 6, nucleotides 867-1200. See also SEQ ID NOs:13 and 14.

An additional object of the present invention is a phenolic acid esterase (i.e., a feruloyl esterase) derived from a previously uncharacterized portion of a Ruminococcus xylanase (See FIG. 1 ). The coding (nucleotides 2164-2895, exclusive of translation start and stop signals) and deduced amino acid sequences (amino acids 546-789) are given in Table 10. See also SEQ ID NOs:15 and 16.

A feruloyl (phenolic acid) esterase is prepared from the anaerobic fungus Orpinomyces PC-2. The coding sequence and deduced amino acid sequences of the mature esterase protein are given in Table 9, and the purification of the Orpinomyces enzyme is described herein below. See also SEQ ID NOs:17 and 18.

Recombinant production of the phenolic (especially ferulic) acid esterases is described. Escherichia coli, Bacillus subtilis , Streptomyces sp., Saccharomyces cerevisiae, Aureobasidium pullulans, Pichia pastoris , Trichoderma, Aspergillus nidulans or any other host cell, including plants, suitable for the production of a heterologous protein can be transfected or transformed with an expression vector appropriate for the chosen host. Compatible combinations of vectors and host cells are well known in the art, as are appropriate promoters to be used to direct the expression of a particular coding sequence of interest. The recombinant host cells are cultured under conditions suitable for growth and expression of the phenolic acid esterase, and the recombinant esterase is then collected or the recombinant host cells in which the esterase has been produced are collected. The coding sequence of the esterase can be operably linked to a nucleotide sequence encoding a signal peptide which is known in the art and functional in the desired host cell if secretion of the esterase into the culture medium is desired. In that case, the culture medium serves as the source of esterase after growth of the host cells.

It is recognized by those skilled in the art that the DNA sequences may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences which encode a phenolic acid esterase polypeptide having a specifically exemplified amino acid sequence are included in this invention, including DNA sequences encoding them having an ATG preceding the coding region for the mature protein and a translation termination codon (TAA, TGA or TAG) after the coding sequence.

Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the phenolic acid esterase polypeptide coding sequences which will not significantly change activity of the amino acid sequences of the polypeptides which the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of a phenolic acid esterase. The skilled artisan will understand that the amino acid sequence of an exemplified phenolic acid esterase polypeptide and signal peptide(s) can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode functional equivalents to the polypeptides defined by the amino acid sequences given herein or an amino acid sequence of greater than 40% identity thereto and having equivalent biological activity. All integer percents between 40 and 100 are encompassed by the present invention. DNA sequences having at least about 75% homology to any of the ferulic acid esterases coding sequences presented herein and encoding polypeptides with the same function are considered equivalent to thereto and are included in the definition of “DNA encoding a phenolic acid esterase.” Following the teachings herein, the skilled worker will be able to make a large number of operative embodiments having equivalent DNA sequences to those listed herein.

Feruloyl esterase proteins are characterized by at least a portion having from at least about 40% amino acid sequence identity with an amino acid sequence as given in SEQ ID NO:18, amino acids 227 to 440 (within the feruloyl esterase protein of Orpinomyces PC-2. All integer percent identities between 40 and 100% are also contemplated. Similarly, feruloyl esterase proteins can have from about 40% to about 100% identity with an amino acid sequence from the group comprising amino acids 581 to 789 of SEQ ID NO:16, amino acids 845 to 1075 of SEQ ID NO:12, amino acids 69 to 286 of SEQ ID NO:14, amino acids 69 to 307 of SEQ ID NO:14, and amino acids 69 to 421 of SEQ ID NO:14. Specifically exemplified feruloyl esterases are characterized by amino acid sequences from the group comprising amino acids 227 to 440 of SEQ ID NO:18, amino acids 581 to 789 of SEQ ID NO:16, amino acids 845 to 1075 of SEQ ID NO:12, amino acids 69 to 286 of SEQ ID NO:14, amino acids 69 to 307 of SEQ ID NO:14, and amino acids 69 to 421 of SEQ ID NO:14. Feruloyl esterase proteins include those having the following amino acid sequences: SEQ ID NO:18, amino acids 1 to 530; SEQ ID NO:12, amino acids 795 to 1077; SEQ ID NO:16, amino acids 546 to 789; SEQ ID NO:14, amino acids 20 to 286; SEQ ID NO:14, amino acids 20 to 307; and SEQ ID NO:14, amino acids 20 to 421.

Specifically exemplified nucleotide sequences encoding the feruloyl esterase proteins include the following: SEQ ID NO:17, nucleotides 1 to 1590; SEQ ID NO:11, nucleotides 2582-3430; SEQ ID NO:15, nucleotides 2164 to 2895; SEQ ID NO:13, nucleotides 158 to 958; SEQ ID NO:13, nucleotides 158 to 1021; SEQ ID NO:13, nucleotides 158 to 1363.

The phenolic acid esterase coding sequences, including or excluding that encoding a signal peptide, can be used to express a phenolic acid esterase in recombinant fungal host cells or plant cells as well as in bacteria, including without limitation, Bacillus spp., Streptomyces sp. and Escherichia coli . Any host cell in which the signal sequence is expressed and processed may be used. Preferred host cells are Aureobasidium species, Aspergillus species, Trichoderma species and Saccharomyces cerevisiae , as well as other yeasts known to the art for fermentation, including Pichia pastoris [See, e.g., Sreekrishna, K. (1993) In: Industrial Microorganisms: Basic and Applied Molecular Genetics , Baltz, R. H., et al. (Eds.) ASM Press, Washington, D.C. 119-126]. Filamentous fungi such as Aspergillus, Trichoderma, Penicillium, etc. are also useful host organisms for expression of the DNA of this invention. [Van den Handel, C. et al. (1991) In: Bennett, J. W. and Lasure, L. L. (Eds.), More Gene Manipulations in Fungi, Academy Press, Inc., New York, 397-428].

FIG. 1 shows amino acid sequence alignment of the exemplified phenolic acid esterases. Sequences are xylanase Z [XynZ_Clotm, Grépinet et al. (1988) supra], xylanase Y [XynY_Clotm, Fontes et al. (1995) supra] of C. thermocellum , xylanase A (XynA_Rumin) of a Ruminococcus sp, and a hypothetical 44-kDa protein of E coli (Genbank Accession Number P31471) (SEQ ID NO:19). Amino acid numbering was the same as in the databases. Dots represent gaps introduced to optimize alignment, and are treated as mismatched in calculations of sequence relatedness (similarity or identity). The partial amino acids are derived from SEQ ID NO:20, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:19 and SEQ ID NO:18.

FIG. 2 shows the domain organizations of two cellulosomal components, xylanase Y and xylanase Z of C. thermocellum.

FIG. 3 illustrates the results of Superose 6 gel filtration of proteins eluted from Avicel adsorption of C. thermocellum culture supernatant. Fractions (0.5 ml) were collected and assayed for protein and feruloyl esterase activity. Molecular mass standards (Sigma Chemical Company, St. Louis, Mo.) including blue dextran (2,000 kDa), catalase (232 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa) were run under identical conditions and their elution positions were indicated.

FIG. 4 presents amino acid sequence alignment of family VI cellulose binding domains. Sequences are xylanase U (XynU_Clotm), xylanase V (XynV_Clotm) (Fernandes et al., 1998, Genbank Accession Number AF047761), and xylanase Z [XynZ_Clotm, Grépinet et al. (1988) supra] of C. thermocellum and xylanase A [XynA Closr, Sakka et al. (1993) Biosci. Biotech. Biochem . 57:273-277; Sakka et al. (1996) Ann. N.Y. Acad Sci . 782:741-751] of C. stercorarium . The sequences presented are portions of those sequences presented in SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:24.

FIG. 5 shows the results of SDS-PAGE analysis of the C. thermocellum XynZ ferulic acid esterase+cellulose binding domain (FAE/CBD) over-expressed in E. coli . Lane M, low range protein standard markers (Bio-Rad Laboratories, Hercules, Calif.) including phosphorylase B (97.4 kDa), serum albumin (66.2), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa); lane 1 , E. coli cell free extract; lane 2, heat-treated cell free extract.

FIGS. 6A and 6B, respectively, illustrate the effects of temperature and pH on feruloyl esterase activity of the C. thermocellum XynZ FAE/CBD. Buffer used for evaluating temperature effects was 50 mM sodium citrate, pH 6.0. Assays mixtures with a pH range from 2 to 10 were formulated by using a universal phosphate buffer system.

FIG. 7 illustrates the results of SDS-PAGE analysis of the purified feruloyl esterase from the culture supernatant of Orpinomyces sp. strain PC-2 (lane 1); molecular mass markers are in lane 2.

FIGS. 8A and 8B show the temperature and pH activity profiles, respectively, of the Orpinomyces sp. strain PC-2 feruloyl esterase.

FIG. 9 shows alignment of protein sequences exhibiting homology to the Orpinomyces feruloyl esterase. Sequences are: faea_orpin, Orpinomyces sp. strain PC-2 FaeA; xyna_rumin, xylanase from Ruminococcus sp. (Genbank Accession Number S58235); yiel_ecoli hypothetical 44kDa protein from E. coli (Genbank Accession Number P31471); xyny_clotm, xylanase Y from C. thermocellum (Genbank Accession Number P51584); xynz_clotm, xylanase Z from C. thermocellum (Genbank Accession Number M22624); dppv_asprf, dipeptidyl peptidase from A. fumigatus (Genbank Accession Number L48074) (SEQ ID NO:20). The partial sequences are taken from. SEQ ID NO:18, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:20.

FIG. 10 is a schematic diagram of the faeA gene from Orpinomyces PC-2.

FIG. 11 illustrates the synergistic effects of the Orpinomyces FaeA and XynA on the release of ferulic acid from wheat bran as substrate.

The amino acids which occur in the various amino acid sequences referred to in the specification have their usual three- and one-letter abbreviations routinely used in the art: A, Ala, Alanine; C, Cys, Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe, Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, Ile, Isoleucine; K, Lys, Lysine; L, Leu, Leucine; M, Met, Methionine; N, Asn, Asparagine; P, Pro, Proline; Q, Gln, Glutamine; R, Arg, Arginine; S, Ser, Serine; T, Thr, Threonine; V, Val, Valine; W, Trp, Tryptophan; and Y, Tyr, Tyrosine.

Additional abbreviations used in the present specification include the following: aa, amino acid(s); bp, base pair(s); CD, catalytic domain(s); GCG, Genetics Computer Group, Madison, Wiss.; CMC, carboxymethyl cellulose; FPase, filter paper-ase; HMWC, high-molecular weight complex(es); IPTG, isopropyl-β-D-thiogalactoside; OSX, oat spelt xylan; ORF, open reading frame; RBB, remazol brilliant blue; pfu, plaque forming units, FAXX, (0-{5-0-[(E)-feruloyl]-α-L-arabinofuranosyl}-(1→3)-0-β-D-xylopyranosyl-(1→4)-D-xylopyranose.

In the present context, plant-derived means material from a plant or plant part. The material can be seeds, grain, foliage, stems, woody or fibrous material, and the like.

In this application derived from a microorganism means that a nucleic acid segment or a protein is purified or taken directly from that microorganism or a culture thereof; A protein produced by genetically engineered (recombinant) using DNA sequence information from the microorganism is also considered derived from that microorganism.

Genes encoding feruloyl esterase (faeA) have been cloned from Aspergillus niger and Aspergillus tubingensis and the deduced amino acid sequences bear close similarity to lipases [de Vries et al. (1997) Appl. Environ. Microbiol . 63:4638-4644]. Expression of these gene products is regulated by the xlnR gene product [van Peij et al. (1998) Appl. Environ. Microbiol . 64:3615-3619]. Other genes include the xylD gene from Pseudomonas fluorescens subsp. cellulosa, the gene product of which has a higher specificity for acetyl groups than feruloyl groups [Ferreira et al. (1993) Biochemical J . 294:349-355] and two genes from Butyrivibro fibrisolvens termed cinA and cinB [Dalrymple and Swadling (1997) Microbiology 143:1203-1210; Dalrymple et al. (1996) FEMS Microbiol. Lett . 143:115-120]. These genes are believed to be regulated by the cinR gene product which may itself be regulated by FAXX [Dalrymple and Swadling (1997) supra]. Esterase activity has also been studied in Streptomyces olivochromogenes [Faulds and Williamson (1991) J. Gen. Microbiol . 137:2339-2345 ], Schizophyllum commune [MacKenzie and Bilous (1988) Appl. Environ. Microbiol . 54:1170-1173 ], Penicillium pinophillum [Castanares and Wood (1992) Biochem. Soc. Trans . 20:275S], and Fibrobacter succinogenes [McDermid et al. (1990) Appl. Environ. Microbiol . 56:127-132].

As described herein, feruloyl esterases are found as part of xylanases from the Clostridium thermocellum cellulosome or as an individual enzyme, for example, from Orpinomyces sp. PC-2. Xylanases Y and Z from C. thermocellum are composed of a xylanase domain, a linker domain, and other domains as well as a domain to which no function has been assigned. We found partial sequence homology between these enzyme and the feruloyl esterase of Orpinomyces in the region of the previously unknown domains and demonstrated that these domains indeed encode feruloyl esterases. Herein, we also report the purification, cloning, and partial characterization of the feruloyl esterase from Orpinomyces sp. strain PC-2.

Anaerobic fungi produce high levels of phenolic esterases [Bomeman and Akin (1990) In: Microbial and Plant Opportunities to Improve Lignocellulose Utilization by Ruminants. D. E. Akin, L. G. Ljungdahl, J. R. Wilson, and P. J. Harris (Eds.). Elsevier Science Publishing Co. New York, pp. 325-340] and two feruloyl esterases of the anaerobic fungus Neocallimastix MC-2 were purified and characterized [Borneman et al. (1992) Appl. Environ. Microbiol . 58:3762-3766]. A cDNA coding for a feruloyl esterase (FaeA) of the anaerobic fungus Orpinomyces PC-2 was cloned and sequenced by the present inventors. FASTA and BLAST searches showed that the catalytic domain of the Orpinomyces FaeA was over 30% identical to sequences coding for unknown domains (UD) in the databases including the carboxy terminal region of XynY Fontes et al. (1995) supra], the amino terminal region of XynZ [Grépinet et al. (1988) supra], a hypothetical polypeptide of E. coli (Genbank Accession Number P31471), and the carboxy terminal region of a Ruminococcus xylanase [Genbank Accession No. S58235] (FIG. 1 ). No function had been previously assigned to the sequences homologous to the Orpinomyces FaeA. XynY consists of multiple domains including a family F xylanase domain, followed by a putative thermostability domain, a dockerin, and the UD [Fontes et al. (1995) supra]. Similarly, XynZ is also multi-domain enzyme containing the UD, a family VI cellulose binding domain, a dockerin, and a family 10 xylanase domain [Grépinet et al. (1988) supra; Tomme et al. (1995) In: Enzymatic Degradation of insoluble Carbohydrates . J. N. Saddler, M. H. Panner (Eds.), ACS Symposium Series, American Chemical Society, Washington, D.C., pp. 142-163]. Both XynY and XynZ are believed to be components of the cellulosome (FIG. 2 ). The Orpinomyces FaeA together with those homologous sequences, however, failed to show significant homology to the recently published feruloyl esterases (FaeA) of Aspergillus niger and A. tubingensis [de Vries et 5 al. (1997) supra]. The sequence analysis implies that a new type of feruloyl esterase is encoded by the Orpinomyces cDNA and the homologous sequences described above.

We have determined that C. thermocellum produces feruloyl esterase activity under the conditions when the cellulosome production is induced. The bacterium was cultivated on low concentration (0.2%, w/v) of Avicel, and under this growth condition, most of the substrate was consumed and cellulosomes released into culture medium, as indicated by the activities on Avicel and xylan (Table 2). Most of the feruloyl esterase activity (97.9%) was found in the culture medium (Table 2). It is well documented that cellulosomes of C. thermocellum are readily adsorbed to cellulose [Morag et al. (1992) Enzyme Microb. Technol . 14:289-292; Choi and Ljungdahl (1996) Biochemistry 35:4897-4905], and thus Avicel adsorption was used to assess association of the feruloyl activity with cellulosomes. As shown in Table 2, 97.1% of total feruloyl activity was removed from the culture medium by Avicel treatment, even higher than the percentages of cellulase (80.5%) and xylanase (73.3%) activities removed. These data indicate that feruloyl esterases produced by C. thermocellum possess cellulose-binding ability through either a cellulose-binding domain or the cellulosomes. XynZ has a family VI cellulose binding domain [Grépinet et al. (1988) supra; Tomme et al. (1995) supra] and a docking domain between the CBD and the dockerin, whereas XynY contains a docking domain.

Cellulosomes eluted from Avicel adsorption were analyzed by gel filtration chromatography using a Superose 6 column to assess the sizes of proteins containing feruloyl esterase activity in the native state. The majority of the proteins were eluted in fractions containing molecules with sizes around 2.0 million daltons (FIG. 3 ), characteristic of cellulosomes eluted from gel filtration [Choi and Ljungdahl (1996) supra]. Feruloyl esterase activity in the fractions correlated well with fractions of cellulosomes. No activity was found in fractions with protein molecules less than 200 kDa, indicating that feruloyl esterase activity resides in the cellulosome.

The UD coding region of XynY and various regions of XynZ were over-expressed in E. coli using the pRSET system (Invitrogen, Carlsbad, Calif.). Constructs spanning the XynY UD sequence, XynZ UD alone, and UD plus the CBD sequence in PRSET gave high levels of feruloyl esterase activity whereas cell-free extracts of E. coli harboring the pET-21 b recombinant plasmid failed to hydrolyze FAXX. Constructs with 20 and 40 amino acid residues deleted from the C-terminus of the XynZ UD did not hydrolyze FAXX, indicating that XynZ sequence from the end of the signal peptide up to amino acid 288 was required to form an active feruloyl esterase. The heterologous protein band of the UD constructs without IPTG induction on SDS-PAGE analysis reached 40-50% of total protein. Both growth rates and levels of feruloyl activity of the constructs with the XynY and XynZ sequences were lower with IPTG induction than without induction. Without wishing to be bound by theory, it is believed that low level of T7 polymerase in E. coli BL21 (DE3) strain was ideal for the expression of the inserted genes in pRSET B, and over-expression of T7 polymerase gene by IPTG induction resulted in toxic levels of feruloyl esterase production.

Amino acid residues 328 to 419 of XynZ were homologous to two repeated CBDs of C. stercorarium XynA [Sakka et al. (1993) supra; Sakka et al. (1995) supra] (FIG. 4 ). This domain has been recently classified as a family VI CBD [Tomme et al. (1995) supra]. Constructs containing the UD alone and both the UD plus the putative CBD of XynZ were purified from recombinant E. coli cultures. The majority of feruloyl esterase activity of the polypeptide containing both domains was removed by Avicel and acid swollen cellulose adsorption but not with the UD alone, indicating that strong cellulose binding capability resides in the family VI cellulose binding domain of XynZ. Cellulose-binding ability was confirmed with native gel retardation analysis.

The polypeptide of the Fae domain plus CBD (FAE/CBD) has been purified from E. coli cell free extract to almost homogeneity after a single step of heating at 70° C. for 30 min. Over 200 milligrams of the FAE/CBD were obtained from 2.5 gram crude proteins (Table 3). The purified FAE/CBD had a mass of 45 kDa as revealed by SDS-PAGE (FIG. 6 ), consistent with the calculated size (46.5 kDa). This size was also consistent with what was seen on gel filtration. There was no evidence for aggregation of the recombinant polypeptides produced in E. coli.

The purified protein had a Vmax of 13.5 μmol ferulic acid released min-1 mg-1 and Km of 3.2 mM using FAX 3 as substrate. The enzyme had the highest specific activity toward FAXX, but it was almost as active as toward FAX 3 (Table 4). The protein released low levels of ferulic acid from ethyl ferulic acid, ground wheat bran, and Coastal Bermuda grass and p-coumaroyl acid from PAX 3 and ethyl-p-coumaroyl acid. The protein lacked activity toward CMC, Avicel, p-nitrophenyl (pNP)-arabinopyranoside, pNP-glucopyranoside, pNP-xylopyranoside, and pNP-acetate.

The recombinant FAE/CBD enzyme had high levels of activity between pH 3.8 and 7 and temperatures between 37 and 65° C. (FIG. 6 ). The FAE/CBD was stable at temperatures up at 65° C. for 6 hours.

In order to understand how microorganisms breakdown plant cell wall material, we chose to study enzymes from Clostridium thermocellum . In particular, XynY and XynZ from this organism were originally thought to contain a xylanase domain and second domain of unknown function. We have now demonstrated that the function of this domain is that of a feruloyl esterase, which is functional in the cellulosome or as a free protein. Feruloyl esterases are important for the complete degradation of plant cell wall material. These enzymes are produced by several organisms, but they have not been found in a bifunctional enzyme.

A feruloyl esterase from Orpinomyces PC-2 was purified and internal fragments of the enzyme were used to screen the Orpinomyces PC-2 cDNA library. A partial clone was sequenced and showed homology to XynZ. A BLAST analysis showed that this enzyme, along with XynY, had domains of unknown function.

The high temperature stability of the enzyme is surprising because no other thermophilic feruloyl esterases have been reported until the present disclosure of the C. thermocellum thermotolerant feruloyl esterases. The Orpinomyces PC-2 enzyme has substrate specificity for both feruloyl and p-coumaroyl esterified substrates. The clostridial enzymes are the first from bacteria to have such a dual role. Although the Orpinomyces enzyme is not a true p-coumaroyl esterase, no p-coumaric acid esterases have been found in bacteria to date.

Applications for the enzymes of the present invention include producing ferulic acid from wheat bran or agricultural byproducts, using the enzyme to treat grasses or other plant materials or other plant materials used in the pulp and paper industries, feed processing, and as a food additive. These thermostable enzymes have advantages over other enzymes since they are economically and easily purified, they have high temperature optima, good thermostability, and they are stable over a wide range of pH values.

Feruloyl esterases and xylanase act synergistically to the release of ferulic acid and reducing sugars from lignocellulosic material [Borneman et al. (1993) supra]. In C. thermocellum XynY and XynZ, we hypothesize that this is more efficient due to the incorporation of both enzymes into one. We believe there is a multicutting event catalyzed by these enzymes much like the multicutting event in the cellulosome itself which leads to more efficient hydrolysis of plant cell wall material. The substrate, arabinoxylan could be passed from one active site to another, which would eliminate the process of each of two enzymes having to bind to the substrate and then release it for the other enzyme to attack.

XynY and XynZ are enzymatic components of the Clostridium thermocellum cellulosome. These components have a multi-domain structure which includes a dockerin domain, a catalytic xylanase domain, and a domain of unknown function. The previously unknown domains in XynY and XynZ have been found to have phenolic esterase activity. These domains have some amino acid homology to that of a phenolic esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Secondly, purified cellulosomes from C. thermocellum hydrolyze (O-{5-O-[(E)-feruloyl]-(-L-arabinofuranosyl}-(1(3)-O-(-D-xylopyranosyl-(1(4)-D-xylopyranose) (FAXX) and {5-O-[(E)-feruloyl]-[O-(-D-xylopyranosyl-(1(2)]-O-(-L-arabinofuranosyl-[1(3]}-O-(-D-xylopyranosyl-(1(4)-D-xylopyranose (FAX 3 ) yielding ferulic acid as a product, thus indicating the presence of a phenolic acid esterase. Intracellular and extracellular fractions lacking cellulosomes had insignificant amounts of phenolic acid esterase activity which confirmed that the activity resided with the cellulosome. The final proof was obtained by cloning the domains of XynY and XynZ into Escherichia coli . The domains were expressed and found to possess phenolic acid esterase activities with FAXX and FAX 3 as substrates.

Nucleotides corresponding to regions of DNA encoding amino acids in XynZ (Genbank Accession Number M22624) from 20-421 and in XynY (Genbank Accession Number X83269) from 795-1077 were overexpressed in E. coli using the pET and pRSET systems respectively. The XynZ sequence will henceforth be referred to as XynZ FAE/CBD since it incorporates the family VI CBD, and the XynY protein is XynY FAE since it only contains a catalytic domain. The cell free extracts containing the expressed proteins each hydrolyzed FAXX with release of ferulic acid (FA) which suggests that these proteins are feruloyl esterases. The expressed protein from the construct containing XynY FAE had a molecular weight of 31 kDa, consistent with the sequence data. Constructs containing XynZ FAE/CBD produced a protein with a molecular mass of 45 kDa as analyzed by SDS-PAGE. The protein was expressed without IPTG induction at a level of 8% of the total protein. Levels of feruloyl esterase activity of the constructs with the XynY FAE and XynZ FAE/CBD sequences were lower with IPTG induction than without induction. Since these proteins had similar sequences and similar function coupled with the fact that XynZ had higher expression levels than XynY, we decided to focus our attention on XynZ and subsequent experiments will refer to that protein.

Constructs were made which corresponded to proteins with amino acids from the original C. thermocellum XynZ sequence of 20-307 (FAE287), 20-286 (FAE) and 20-247 (FAE227) (with reference to SEQ ID NO:14 and FIG. 2 ). FAE287 is missing the CBD, but contains a proline rich linker which separates the CBD from the FAE domain while FAE does not contain this linker. When these constructs were expressed in E. coli in the same manner as XynZ FAE/CBD, they both exhibited feruloyl esterase activity. Thus, the removal of the 114 amino acids of the CBD did not have a detrimental effect on the activity. XynZ FAE/CBD bound to acid swollen cellulose very weakly, while the other constructs missing the CBD did not bind acid swollen cellulose at all. FAE227 was an inactive but expressed enzyme. Neither the CBD nor the linker is necessary for activity, but amino acids 247-266 are necessary for generation of an active enzyme. Since neither the linker region nor the CBD is necessary for activity, we used the smallest construct which still retained activity, FAE, for subsequent experiments.

The XynZ FAE/CBD polypeptide was purified from E. coli cell free extract after a single step of heat treatment at 70° C. for 30 min. Over 200 mg of the XynZ FAE/CBD were obtained from 2.5 gram of crude protein (Table 3). The purified XynZ FAE/CBD had a mass as stated previously of 45 kDa as revealed by SDS-PAGE (FIG. 5 ), consistent with the calculated size (46.5 kDa). There was no evidence for aggregation of the feruloyl esterase produced in E. coli , and SDS-PAGE gels showed that protein which was removed from the cell free extract by centrifugation had no insoluble protein which could be attributed to inclusion bodies.

The purified protein had a Vmax of 12.5 μmol ferulic acid released min-1 mg-1 and Km of 5 mM using FAX3 as substrate. The enzyme had the highest specific activity towards FAXX but was almost as active toward FAX3 (Table 4). The protein was able to release low levels of FA from ethyl ferulic acid, ground wheat bran, and Coastal Bermuda grass and p-coumaric acid (PCA) from PAX3 and ethyl-p-coumarate. The protein lacked activity toward CMC, Avicel, p-nitrophenyl (pNP)-arabinopyranoside, pNP-glucopyranoside, pNP-xylopyranoside, and pNP-acetate. Isoelectric focusing gel electrophoresis showed that the protein had a pI of 5.8.

The FAE polypeptide of XynZ was also expressed and purified to homogeneity. A purification scheme is shown in Table 3B. The protein was expressed in a manner similar to that for XynZ FAE/CBD. The heat treatment step also resulted in 200 mg of protein, but the protein was not pure. An additional step involving gel filtration resulted in a pure enzyme with a Vmax of 28.2 μmol ferulic acid released min-1 mg-1 and Km of 10.5 mM using FAX3 as substrate. FAE was inhibited by ferulic acid but not by xylose or arabinose. The FAE had a temperature optimum between 30° and 70° C. (FIG. 6A) and had high level activity between pH 4 and 7 (FIG. 6B) The enzyme was stable at temperatures up at 70° C. for 6 hours, and in a similar experiment, FAE/CBD also was stable at 70° C. At 80° C., the relative activity of FAE decreased to around 50% after three hours of incubation, and most of the relative activity was destroyed after 1 hour of incubation at 90° C.

Anaerobic microorganisms do not readily degrade lignin, but are able to solubilize it. Anaerobic fungi are able to solubilize but not metabolize lignin, and it is suggested that the released lignin was carbohydrate linked [McSweeney et al. (1994) Appl. Environ. Microbiol . 60:2985-2989]. The data herein indicate that feruloyl esterases are responsible for lignin solubilization. Most studies of the cellulosome of C. thermocellum has been directed toward its celluloytic activity. It also has xylanases which we have shown are bifunctional enzymes with feruloyl esterase activity. The cellulosome should be efficient in the degradation of arabinoxylan. It has been previously shown that Clostridium xylanolyticum released aromatics into the culture medium when grown on lignocellulosic material [Rogers et al. (1992) International Biodeterioration & Biodegradation 29:3-17].

XynY and XynZ each contain a glycosyl hydrolase family 10 catalytic domain in addition to the FAE catalytic domain. The xylanase domain of XynZ has been well studied, that construct has been crystallized, and the three dimensional structure solved [Dominguez et al. (1995) Nat. Struct. Biol . 2:569-576; Souchon et al. (1994) J. Mol. Biol . 235:1348-1350]. In general, xylanases are thought to be sterically hindered by groups substituted on the xylan backbone. Feruloyl esterase and xylanase have been shown to act synergistically for the release of ferulic acid and reducing sugars from lignocellulosic material [Bomeman et al. (1993) supra]. In XynY and XynZ we hypothesize that this event has been made more efficient by the incorporation of both FAE and xylanase catalytic domains into one enzyme. Without wishing to be bound by theory, we believe that there is a multicutting event catalyzed by these enzymes much like the multicutting event in the cellulosome itself which leads to more efficient hydrolysis of plant cell wall material. Bifunctional enzymes like XynY and XynZ form a dumbbell-like shape which attacks the arabinoxylan polysaccharide and the substrate is passed from one active site to another, eliminating the relatively inefficient two enzyme process in which one has to bind to the substrate and then release it for the other enzyme to attack. The existence of multidomain enzymes such as the sea whip coral peroxidase-lipoxygenase [Koljak et al. (1997) Science 277:1994-1996] and a xylanase-β(1,3-1,4)-glucanase from Ruminococcus flavifaciens [Flint et al. (1993) J. Bacteriol . 175:2943-2951] suggests an evolutionary importance of having two or more catalytic domains in one enzyme. XynZ contains a contains a family VI CBD, which does not bind cellulose significantly. However, representatives of CBDs of this family usually efficiently bind xylan. The CBD of XynZ may participate in a tight association of the catalytic domains with the substrate. This is consistent with the higher Km of FAE as compared to that of XynZ FAE/CBD.

Both FAE/CBD and FAE are highly thermostable. They are active against both feruloyl and p-coumaroyl esterified substrates, and they represent the first FAE from bacteria to hydrolyze p-coumaroyl esters. The high Km of FAE versus XynZ FAE/CBD indicates that the CBD is important in binding the substrate before enzyme catalysis.

The FAE domains of XynZ and XynY are homologous to each other and to the Orpinomyces FaeA. The Orpinomyces FaeA, together with those homologous sequences, however, failed to show significant homology to the recently published feruloyl esterases (FaeA) of Aspergillus niger and A. tubingensis [de Vries et al. (1997) supra] as well as CinA and CinB from Butyrivibrio fibrisolvens [Dalrymple et al. (1996) FEMS Microbiol. Lett . 143:115-120; Dalrymple and Swadling (1997) Microbiology 143:1203-1210] and XylD from Pseudomonas fluorescens subsp. cellulosa [Ferreira et al. (1993) Biochemical Journal 294:349-355]. The sequence analysis implies that a new type of feruloyl esterase is encoded by the Orpinomyces gene and the homologous C. thermocellum sequences described above. The Orpinomyces FaeA, and the FAE domains of XynZ and XynY were also shown to be homologous to a hypothetical polypeptide of E. coli (Genbank Accession Number P31471) and the carboxy terminal region of a Ruminococcus sp. xylanase earlier designated as a UD [Genbank Accession Number S58235]. No function had been assigned to those sequences of E. coli and Ruminococcus. Without wishing to be bound by theory, the present inventors believe that these sequences also encode feruloyl esterases and that the Ruminococcus xylanase is also bifunctional. Ruminococcus has been shown to produce FAE activity [McSweeney et al. (1998) Anaerobe 4:57-65], and another Ruminococcus xylanase has been shown to be a bifunctional enzyme with xylanase and acetyl xylan esterase activity [Kirby et al. (1998) Biochemical Society Transactions 26:S169]. No feruloyl esterase activity has been observed in E. coli . The gene from E. coli may encode a dipeptidase instead, because homology exists between a dipeptidase from Aspergillus fumigatus and feruloyl esterases. The data suggest a common ancestral gene encoding feruloyl esterases from Orpinomyces, C. thermocellum , and Ruminococcus.

Applications for the phenolic acid esterase enzymes of the present invention, especially the feruloyl esterases, include producing ferulic acid from wheat bran or agricultural byproducts, using the enzyme to treat grasses, grains or other plant materials which are used in the pulp and paper industry, feed processing, and as a food additive. These thermostable enzymes have advantages over other enzymes because they are easy to purify, have high temperature optima and are stable over a wide pH range.

Any of the feruloyl esterases as described (see also U.S. patent application Ser. No. 09/390,224, filed Sep. 3, 1999, incorporated by reference herein) can be used as food or feed supplements. Other phenolic acid or feruloyl esterases known to the art can also be used in the methods and compositions of the present invention. For example, esterases isolated after production from a naturally occurring microbial strain or through recombinant production using nucleic acids ultimately derived from a naturally occurring strain are contemplated. Naturally occurring microbes which produce phenolic acid esterases and/or feruloyl esterases include, without limitation, strains of Aureobasidium, Trichoderma, Aspergillus, Bacillus, Streptomyces, Penicillium, Neocallimastix, and Humicola, as well known in the art.

Xylanases are also available from a variety of sources including but not limited to, Orpinomyces PC-2 , Aureobasidium pullulans , Neocallimastix, Clostridium, Bacillus, Streptomyces, Thermotoga, Talaromyces, Caldocellum, Thermonospora, ruminent bacteria and fungi, among many others well known to the art.

When a grain or other plant-derived food or feed component having a substantial non-starch polysaccharide content is used, the energy source availability can be increased by treatment with a feruloyl esterase and a xylanase at a ration of 1 to 200 U/kg for each enzyme, desirably about 10 to about 50 U/kg feed or food. As shown in Table 12, there appears to be a synergism between xylanase and feruloyl esterase.

Food or feed can be supplemented or treated with the feruloyl esterase and xylanase to improve nutrition and energy source availability for humans, poultry (e.g., chickens, turkeys, ducks, geese, and other fowl), swine, sheep, cattle, horse, goats, fish (including but not limited to salmon, catfish, tilapia and trout) and shellfish, especially shrimp, and other farmed animals.

Food or feed ingredients which are improved by treatment with feruloyl esterase and xylanase include, without limitation, wheat, rye, barley, oats, corn, rice, soybean, millet, sorghum, grasses, legumes and other pasture and forage plants. Fresh or dry feed or food components can be treated with a liquid comprising the xylanase and phenolic acid esterase so that the particles of the food or feed are coated with the enzymes. Similarly, wet or dry enzyme compositions can be added to a liquid food or feed composition so that the ratio of enzymes to dry weight or plant material is as taught herein.

The present inventors have demonstrated the usefulness of feruloyl esterase as an animal feed additive, as described in Example 7 below. Wheat represents a potential energy source in poultry and swine or other rations, for example, but it is frequently avoided because of its low energy value relative to corn. The lower energy availability is due to the presence of a significant amount of non-digestible fiber or non-starch polysaccharide (NSP). In addition to NSP being unavailable for energy, it also acts as an anti-nutritional factor and reduces digestibility of other components of the diet. The availability of fiber-degrading enzymes that can be added to wheat diets has increased interest in the use of wheat and other grains for poultry and swine rations. Three day old broiler chicks were fed an energy-deficient diet or the same diet supplemented with Orpinomyces or other feruloyl esterase protein, a xylanase A (for example from Orpinomyces or from Aureobasidium pullulans for example [see U.S. Pat. No. 5,591,619, Li et al., 1997]) or a combination of these enzymes. See Table 11 for composition of the basal diet and Example 7. The results of this feeding experiment are summarized in Table 12. Weight gain and feed efficiency in the control group is set to 100. Feruloyl esterase alone did not significantly affect weight gain. Birds grew slightly slower (97% of control) and required slightly more feed per unit of gain than the control, unsupplemented group. Xylanase alone improved growth rate 7% and feed efficiency 5%. The combination of xylanase and feruloyl esterase showed the greatest response, with a 15% improvement in growth rate and a 10% improvement in efficiency over that in the control group.

Besides promoting increased nutrient availability, feruloyl esterase can be used to improve the ferulic acid availability in a food or feed. Ferulic acid has antioxidant activity, and it can be made available through feruloyl esterase treatment of a foodstuff. Ferulic acid is an antioxidant, and accordingly, there is interest in its use to promote general health, to act as an anti-tumor agent and as an anti-aging agent. For example, wheat can be treated with feruloyl esterase, advantageously in combination with xylanase, and be consumed to serve as a ferulic acid supplement, especially in humans. Its consumption then improves the oxidant/antioxidant status and the general health of the consumer.

In addition to plant-derived solid food or feed treated with feruloyl esterase or the combination of feruloyl esterase and xylanase, liquids (beverages, e.g.) can also comprise feruloyl esterase (or feruloyl esterase and xylanase) treated material or soluble products thereof. If the beverage contains the solid foodstuff or feedstuff, enzymes(s) is(are) added at a ratio of from about 1 to 200 units of enzyme per kg, desirably from about 10 to about 50 U/kg of esterase and for xylanase, from about 100 to about 10,000 U/kg dry weight of plant-derived material in the liquid foodstuff or beverage.

A feruloyl esterase described herein, desirably in combination with a cellulase and/or xylanase, for example that from Orpinomyces PC-2, can also be used in the pulping and paper recycling industries. The ratio of the esterase to solids is from about 0.1 to about 200 U/kg dry weight, desirably from about 1 to about 100 U/kg, and advantageously from about 10 to about 50 U/kg.

The feruloyl esterase or combination of feruloyl esterase and xylanase can be formulated as dry materials or as liquid concentrates for subsequent use in combination with a source of plant-derived non-starch polysaccharide or poorly digestible plant fiber material to be treated. Such a formulation can be freeze-dried in the case of a dry material or it can be a liquid concentrate. A liquid formulation can contain from about 100 ug. to about 50 mg/ml of protein. Reducing agents such as cystine dithiothreitol, dethioerythritol or β-mercaptoethanol can be included to prevent enzyme oxidation, and protein stabilizing agents, for example glycerol (0.1% to 10% w/v), sucrose (0.1% to 10% w/v) among others, can be included, or an irrelevant protein such as bovine serum albumin or gelatin, can also be present. Although the esterases of the present invention are stable, a buffering agent can be added to stabilized the pH in the range of about 4.5 to 7.8.

The feruloyl esterase domain of XynZ was highly expressed in E. coli and the esterase comprised 40-50% of the total cell protein. The recombinant esterase of XynZ was purified to almost homogeneity by heat treatment. The protein had a molecular mass of 45 kDa, consistent with the size of the predicted deduced amino acid sequence. Of the substrates tested, the expressed protein had high specific activity towards FAXX and FAX 3 . With FAX 3 as a substrate Km and Vmax values were 3.2 mM and 13.5 μmol ferulic acid released min-1 mg-1 respectively at pH 6.0 at 60° C. Several phenolic esterified substrates were hydrolyzed and the specific activities with those containing feruloyl groups were higher than were those with p-coumaroyl groups confirming that the previously unknown domain of XynZ is a feruloyl esterase. The enzyme released mainly ferulic acid from wheat bran and Coastal Bermuda grass (CBG) with a smaller amount of p-coumaroyl groups released from CBG. This study represents the first demonstration of esterases in the cellulosome of Clostridium thermocellum and of enzymes from the cellulosome with two different activities. The present work also provides a phenolic acid esterase derived from a xylanase from Ruminococcus and as an enzyme produced by Orpinomyces PC-2.

A summary of the purification of FAE from Orpinomyces sp stain PC-2 is presented in Table 7. The Q-Sepharose column separated two peaks of esterase activity. Proteins which eluted in the first peak had higher activity against ethyl-pCA while proteins eluting in the second peak had greater activity against FAXX. These data suggest that a p-coumaroyl esterase eluted in the first peak while the feruloyl esterase eluted in the second. The first peak was not studied further, but the fractions in peak 2 were further purified resulting in a purified enzyme which had an approximate molecular mass of 50 kDa as visualized by SDS-PAGE analysis (FIG. 7 ). There was a decrease in specific activity after the MonoQ step which could not be explained.

Temperature and pH optima experiments showed that the enzyme had a temperature optimum of 50° C. (FIG. 8A) and had activity over a pH range between 5.2 and 8 (FIG. 8 B). The purified enzyme was stable at 4° C. for over 18 months. The purified enzyme was subjected to N-terminal sequencing giving the sequence ETTYGITLRDTKEKFTVFKD (SEQ ID NO:21). The protein was also subjected to internal sequencing which resulted in four peptide fragments (Table 8) which were used to create degenerate PCR primers.

Two of the peptide fragments from the internal amino acid sequencing were used to create degenerative olignucleotide primers which are listed in the materials and methods section. These primers were used to amplify regions of DNA in the Orpinomyces PC-2 cDNA library. A 216 bp PCR product was generated. The PCR product was labeled with digoxygenin-UTP and used as a probe to screen the cDNA library. After screening 50,000 phage, one positive plaque was obtained and its DNA was sequenced using T3 and T7 universal primers. Sequencing using the T3 primer did not reveal any ORFs, however, sequencing using the T7 reverse primer gave the C-terminal end of the gene. Based on the sequence data and restriction fragment analyses, but without wishing to be bound by theory, we have concluded that the faeA gene in this cDNA was truncated and furthermore that the insert comprises multiple genes. These other genes were not studied further. The deduced amino acid sequence of the insert matched the data from the peptide sequencing. The insert had a size of 1074 bp and encoded a protein of 358 amino acids. Since the size of the encoded protein did not match that of the purified enzyme and the N-terminal sequence, including a signal peptide and lack of a start codon, another round of screening was performed using the entire sequence as a probe after digoxygenin labeling. After screening an additional 50,000 phage, one positive clone was obtained which had a size of 1673 bp with the largest open reading frame comprising a protein of 530 amino acids. The sequence of this insert is believed to be an incomplete one since no 5′ UTR was found and the (putative) signal sequence has only four amino acids. Most signal sequences found in hydrolytic enzymes from anaerobic fungi are at least 20 amino acids long. The insert was found to be in a reverse orientation with respect to the lacZ promoter. The upstream lac promoter should direct synthesis of the inserted gene, but no activity was found in lysed E. coli cells harboring the recombinant plasmid. The faeA gene in E. coli was expressed using the pET system (Novagen) in the correct orientation. The recombinant FaeA released ferulic acid from FAXX as well as other substrates which were esterified with phenolic groups. The enzyme had the highest activity against FAXX, which demonstrates that it is a true feruloyl esterase (Table 10). In addition, when the enzyme was incubated with a recombinant xylanase, there was a 80 fold increase in FA released over FaeA alone.

The nucleotide and deduced amino acid sequence of the faeA gene are shown in Table 9. A BLAST analysis of the encoded protein showed homology to several enzymes. These enzymes included domains of unknown function from Xylanase Z and Xylanase Y of Clostridium thermocellum , a domain of unknown function in a xylanase from Ruminococcus spp. and a 44 kDa hypothetical protein from E. coli , and a dipeptidyl peptidase from Aspergillus fumigatus (FIG. 9 ). All proteins had at least 20% identity with the C-termninal 300 amino acids of the protein. The N-terminal part of the enzyme did not show homology to any enzyme in the 3 BLAST analysis and the function of this domain is unknown. Although FAE activity has been demonstrated in the cellulase/hemicellulase complex from Orpinomyces, this protein does not contain a non-catalytic repeated peptide domain (NCRPD). Analysis of C-terminal coding region indicated a typical signature sequence found in lipases and other esterases of GXSXG at residues 341-345 as well as an aspartic acid at residue 403 and a histidine at residue 436 which would make up the catalytic triad. A search of the sequence revealed two N-glycosylation sites at amino acids 300 and 488 (of SEQ ID NO:18) and a 16mer poly A tail in the 3′ UTR.

It will be understood by those skilled in the art that other nucleic acid sequences besides those disclosed herein for the phenolic acid esterases, i.e. feruloyl esterases, will fiction as coding sequences synonymous with the exemplified coding sequences. Nucleic acid sequences are synonymous if the amino acid sequences encoded by those nucleic acid sequences are the same. The degeneracy of the genetic code is well known to the art. For many amino acids, there is more than one nucleotide triplet which serves as the codon for a particular amino acid, and one of ordinary skill in the art understands nucleotide or codon substitutions which do not affect the amino acid(s) encoded. It is further understood in the art that codon substitutions to conform to common codon usage in a particular recombinant host cell is sometimes desirable.

Specifically included in this invention are sequences from other strains of Clostridium and from other microorganisms which hybridize to the sequences disclosed for feruloyl and coumaryl esterases under stringent conditions. Stringent conditions refer to conditions understood in the art for a given probe length and nucleotide composition and capable of hybridizing under stringent conditions means annealing to a subject nucleotide sequence, or its complementary strand, under standard conditions (i.e., high temperature and/or low salt content) which tend to disfavor annealing of unrelated sequences, (indicating about 95-100% nucleotide sequence identity). Also specifically included in this invention are sequences from other strains of Orpinomyces species and other anaerobic fungi which hybridize to the sequences disclosed for the esterase sequences under moderately stringent conditions. Moderately stringent conditions refer to conditions understood in the art for a given probe sequence and “conditions of medium stringency” means hybridization and wash conditions of 50°-65° C., 1×SSC and 0.1% SDS (indicating about 80-95% similarity). Also specifically included in this invention are sequences from other strains of Orpinomyces, from other anaerobic fungi, and from other organisms, including bacteria, which hybridize to the sequences disclosed for the esterase sequences under highly stringent conditions. Highly stringent conditions refer to conditions understood in the art for a given probe sequence and “conditions of high stringency” means hybridization and wash conditions of 65°-68° C., 0.1×SSC and 0.1% SDS (indicating about 95-100% similarity). Hybridization assays and conditions are further described in Sambrook et al. (1989).

A method for identifying other nucleic acids encoding feruloyl esterase- and/or coumaryl esterase-homologous enzymes is also provided wherein nucleic acid molecules encoding phenolic acid esterases are isolated from an anaerobic fungus, including but not limited to Orpinomyces or an anaerobic bacterium, such as Clostridium or Ruminococcus, among others, and nucleic acid hybridization is performed with the nucleic acid molecules and a labeled probe having a nucleotide sequence that includes all or part of a FAE coding sequence as given in Table 5, 6, 9 and/or 10 herein. By this method, phenolic acid esterase genes similar to the exemplified feruloyl and coumaryl esterases can be identified and isolated from other strains of Clostridium or other anaerobic microorganisms. All or part of a nucleotide sequence refers specifically to all continuous nucleotides of a nucleotide sequence, or e.g. 1000 continuous nucleotides, 500 continuous nucleotides, 100 continuous nucleotides, 25 continuous nucleotides, and 15 continuous nucleotides.

Sequences included in this invention are those amino acid sequences which are 40 to 100% identical to the amino acid sequences encoded by the exemplified C. thermocellum strain feruloyl esterase, amino acids proteins truncated from the XynY or XynZ proteins or the Ruminococcus FAE polypeptide or the Orpinomyces PC-2 FAE polypeptide, all specifically identified herein. Sequences included in this invention are also those amino acid sequences which are 40, 50, 60, 70, 75, 80, 85, 90, 95 to 100%, and all integers between 40% and 100%, identical to the amino acid sequences encoded by an exemplified phenolic acid esterase coding sequence and corresponding to or identifying encoded proteins which exhibit feruloyl esterase activity. In comparisons of protein or nucleic acid sequences, gaps introduced into either query or reference sequence to optimize alignment are treated as mismatches. In amino acid sequence comparisons to identify feruloyl esterase proteins, the reference sequence is, desirably, amino acids 227 to 440 of SEQ ID NO:18 (FAE of Orpinomyces PC-2).

It is well-known in the biological arts that certain amino acid substitutions may be made in protein sequences without affecting the function of the protein. Generally, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate, and isoleucine and valine, are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of protein Sequence and Structure , Volume 5, Supplement 3, Chapter 22, pp. 345-352, which is incorporated by reference herein provides frequency tables for amino acid substitutions which can be employed as a measure of amino acid similarity. Dayhoff et al.'s frequency tables are based on comparisons of amino acid sequences for proteins having the same fraction from a variety of evolutionarily different sources.

Techniques and agents for introducing and selecting for the presence of heterologous DNA in plant cells and/or tissue are well-known. Genetic markers allowing for the selection of heterologous DNA in plant cells are well-known, e.g., genes carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin. The marker allows for selection of successfully transformed plant cells growing in the medium containing the appropriate antibiotic because they will carry the corresponding resistance gene. In most cases the heterologous DNA which is inserted into plant cells contains a gene which encodes a selectable marker such as an antibiotic resistance marker, but this is not mandatory. An exemplary drug resistance marker is the gene whose expression results in kanamycin resistance, i.e., the chimeric gene containing nopaline synthetase promoter, Tn5 neomycin phosphotransferase II and nopaline synthetase 3′ non-translated region described by Rogers et al., Methods for Plant Molecular Biology , A. Weissbach and H. Weissbach, Eds., Academic Press, Inc., San Diego, Calif. (1988).

Techniques for genetically engineering plant cells and/or tissue with an expression cassette comprising an inducible promoter or chimeric promoter fused to a heterologous coding sequence and a transcription termination sequence are to be introduced into the plant cell or tissue by Agrobacterium-mediated transformation, electroporation, microinjection, particle bombardment or other techniques known to the art. The expression cassette advantageously further contains a marker allowing selection of the heterologous DNA in the plant cell, e.g., a gene carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin. Assays for phenolic acid esterase and/or xylanase enzyme production are taught herein or in U.S. Pat. No. 5,824,533, for example, and other assays are available to the art.

A DNA construct carrying a plant-expressible gene or other DNA of interest can be inserted into the genome of a plant by any suitable method. Such methods may involve, for example, the use of liposomes, electroporation, diffusion, particle bombardment, microinjection, gene gun, chemicals that increase free DNA uptake, e.g., calcium phosphate coprecipitation, viral vectors, and other techniques practiced in the art. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens , such as those disclosed by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO publication 120,516 (Schilperoort et al.). Inaddition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells.

The choice of vector in which the DNA of interest is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., replication, protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules. The vector desirably includes a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In addition, preferred embodiments that include a prokaryotic replicon also include a gene whose expression confers a selective advantage, such as a drug resistance, to the bacterial host cell when introduced into those transformed cells. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline, among other selective agents. The neomycin phosphotransferase gene has the advantage that it is expressed in eukaryotic as well as prokaryotic cells.

Those vectors that include a prokaryotic replicon also typically include convenient restriction sites for insertion of a recombinant DNA molecule of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories (Richmond, Calif.) and pPL, pK and K223 available from Pharmacia (Piscataway, N.J.), and pBLUESCRIPT and pBS available from Stratagene (La Jolla, Calif.). A vector of the present invention may also be a Lambda phage vector including those Lambda vectors described in Molecular Cloning: A Laboratory Manual, Second Edition , Maniatis et al., Eds., Cold Spring Harbor Press (1989) and the Lambda ZAP vectors available from Stratagene (La Jolla, Calif.). Other exemplary vectors include pCMU [Nilsson et al. (1989) Cell 58:707]. Other appropriate vectors may also be synthesized, according to known methods; for example, vectors pCMU/K b and pCMUII used in various applications herein are modifications of pCMUIV (Nilson et al., supra).

Typical expression vectors capable of expressing a recombinant nucleic acid sequence in plant cells and capable of directing stable integration within the host plant cell include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al. (1987) Meth. in Enzymol . 153:253-277, and several other expression vector systems known to fraction in plants. See for example, Verma et al., No. WO87/0055 1; Cocking and Davey (1987) Science 236:1259-1262.

A transgenic plant can be produced by any means known to the art, including but not limited to Agrobacterium tumefaciens -mediated DNA transfer, preferably with a disarmed T-DNA vector, electroporation, direct DNA transfer, and particle bombardment (See Davey et al. (1989) Plant Mol. Biol . 13:275; Walden and Schell (1990) Eur. J. Biochem . 192:563; Joersbo and Burnstedt (1991) Physiol. Plant . 81:256; Potrykus (1991) Annu. Rev. Plant Physiol. Plant Mol Biol . 42:205; Gasser and Fraley (1989) Science 244:1293; Leemans (1993) Bio/Technology . 11:522; Beck et al. (1993) Bio/Technology . 11:1524; Koziel et al. (1993) Bio/Technology . 11:194; and Vasil et al. (1993) Bio/Technology . 11:1533.). Techniques are well-known to the art for the introduction of DNA into monocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those tissues.

Many of the procedures useful for practicing the present invention, whether or not described herein in detail, are well known to those skilled in the art of plant molecular biology. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.

Monoclonal or polyclonal antibodies, preferably monoclonal, specifically reacting with the phenolic acid esterases of the present invention may be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice , 2d ed., Academic Press, New York.

Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning , Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning , Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol . 218, Part I; Wu (ed.) (1979) Meth. Enzymol . 68; Wu et al. (Eds.) (1983) Meth. Enzymol . 100 and 101; Grossman and Moldave (Eds.) Meth. Enzymol . 65; Miller (ed.) (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles, of Gene Manipulation , University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology ; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) (1985) Nucleic Acid Hybridization , IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods , Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.

Each reference and patent document cited in the present application is incorporated by reference herein to the extent that it is not inconsistent with the present disclosure.

The following examples are provided for illustrative purposes, and is not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention.

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

2

tacatatgcc tgtagcagaa aatccttc 28

3

32

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

3

gaggaagctt ttacatggaa gaaatatgga ag 32

4

29

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

4

tacatatgct tgtcacaata agcagtaca 29

5

29

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

5

taggatccct tgtcacaata agcagtaca 29

6

31

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

6

gaggaagctt ttagttgttg gcaacgcaat a 31

7

29

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

7

gaggaagctt acttccacac attaaaatc 29

8

29

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

8

gaggaagctt agtttccatc cctcgtcaa 29

9

29

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

9

gaggaagctt agtcataatc ttccgcttc 29

10

32

Artificial Sequence

Description of Artificial Sequence

oligonucleotide used in polymerase chain reaction.

10

gaggaagctt aaacgccaaa agtgaaccag tc 32

11

taagaaactt taaaacaccc tttataaaaa tacaaagaat tacaggcaat tatagtgtaa 60

tgtggatttt aactaaaatg gaaggaggaa tgtaattcgt aatagatatt atgatataat 120

ttgtttagag catgcttaag tttatttaaa tttaatttat aaattaaatt aaaaattaaa 180

atttaaaagg aggttgctt atg aaa aac aag aga gtt ttg gca aaa ata acg 232

Met Lys Asn Lys Arg Val Leu Ala Lys Ile Thr

1 5 10

gct ctt gtg gta ttg ctg gga gtg ttt ttt gta tta ccg tca aac ata 280

Ala Leu Val Val Leu Leu Gly Val Phe Phe Val Leu Pro Ser Asn Ile

15 20 25

agt cag cta tat gct gat tat gaa gtg gtt cat gac act ttt gaa gtt 328

Ser Gln Leu Tyr Ala Asp Tyr Glu Val Val His Asp Thr Phe Glu Val

30 35 40

aac ttt gac gga tgg tgt aac ttg gga gtc gac aca tat tta acg gca 376

Asn Phe Asp Gly Trp Cys Asn Leu Gly Val Asp Thr Tyr Leu Thr Ala

45 50 55

gtt gaa aat gaa gga aac aac ggt aca aga ggt atg atg gta ata aat 424

Val Glu Asn Glu Gly Asn Asn Gly Thr Arg Gly Met Met Val Ile Asn

60 65 70 75

cgc tcc agt gcg agt gac ggt gcg tat tcg gaa aaa ggt ttc tat ctc 472

Arg Ser Ser Ala Ser Asp Gly Ala Tyr Ser Glu Lys Gly Phe Tyr Leu

80 85 90

gac ggt ggt gta gaa tac aag tac agt gtt ttt gta aaa cac aac ggg 520

Asp Gly Gly Val Glu Tyr Lys Tyr Ser Val Phe Val Lys His Asn Gly

95 100 105

acc ggc acc gaa act ttc aaa ctt tct gtg tcc tat ttg gat tcg gaa 568

Thr Gly Thr Glu Thr Phe Lys Leu Ser Val Ser Tyr Leu Asp Ser Glu

110 115 120

aca gaa gaa gaa aat aag gaa gta att gca aca aag gat gtt gtg gcc 616

Thr Glu Glu Glu Asn Lys Glu Val Ile Ala Thr Lys Asp Val Val Ala

125 130 135

gga gaa tgg act gag att tcg gca aaa tac aaa gca ccc aaa act gca 664

Gly Glu Trp Thr Glu Ile Ser Ala Lys Tyr Lys Ala Pro Lys Thr Ala

140 145 150 155

gtg aat att act ttg tca att aca acc gac agc act gta gat ttc att 712

Val Asn Ile Thr Leu Ser Ile Thr Thr Asp Ser Thr Val Asp Phe Ile

160 165 170

ttt gac gat gta acc ata acc cgt aaa gga atg gct gag gca aac aca 760

Phe Asp Asp Val Thr Ile Thr Arg Lys Gly Met Ala Glu Ala Asn Thr

175 180 185

gta tat gca gca aac gct gtg ctg aaa gat atg tat gca aac tat ttc 808

Val Tyr Ala Ala Asn Ala Val Leu Lys Asp Met Tyr Ala Asn Tyr Phe

190 195 200

aga gtt ggt tcg gta ctt aac tcc gga acg gta aac aat tca tca ata 856

Arg Val Gly Ser Val Leu Asn Ser Gly Thr Val Asn Asn Ser Ser Ile

205 210 215

aag gcc ttg att tta aga gag ttt aac agt att acc tgt gaa aat gaa 904

Lys Ala Leu Ile Leu Arg Glu Phe Asn Ser Ile Thr Cys Glu Asn Glu

220 225 230 235

atg aag cct gat gcc aca ctg gtt caa tca gga tca acc aat aca aat 952

Met Lys Pro Asp Ala Thr Leu Val Gln Ser Gly Ser Thr Asn Thr Asn

240 245 250

atc agg gtt tct ctt aat cgt gca gca agt att tta aac ttc tgt gca 1000

Ile Arg Val Ser Leu Asn Arg Ala Ala Ser Ile Leu Asn Phe Cys Ala

255 260 265

caa aat aat ata gcc gtc aga ggt cat aca ctg gtt tgg cac agc cag 1048

Gln Asn Asn Ile Ala Val Arg Gly His Thr Leu Val Trp His Ser Gln

270 275 280

aca cct caa tgg ttt ttc aaa gac aat ttc cag gac aac gga aac tgg 1096

Thr Pro Gln Trp Phe Phe Lys Asp Asn Phe Gln Asp Asn Gly Asn Trp

285 290 295

gtt tcc caa tca gtt atg gac cag cgt ttg gaa agc tac ata aaa aat 1144

Val Ser Gln Ser Val Met Asp Gln Arg Leu Glu Ser Tyr Ile Lys Asn

300 305 310 315

atg ttt gct gaa atc caa aga cag tat ccg tct ttg aat ctt tat gcc 1192

Met Phe Ala Glu Ile Gln Arg Gln Tyr Pro Ser Leu Asn Leu Tyr Ala

320 325 330

tat gac gtt gta aat gag gca gta agt gat gat gca aac agg acc aga 1240

Tyr Asp Val Val Asn Glu Ala Val Ser Asp Asp Ala Asn Arg Thr Arg

335 340 345

tat tat ggc ggg gcg agg gaa cct gga tac gga aat ggt aga tct cca 1288

Tyr Tyr Gly Gly Ala Arg Glu Pro Gly Tyr Gly Asn Gly Arg Ser Pro

350 355 360

tgg gtt cag atc tac gga gac aac aaa ttt att gag aaa gca ttt aca 1336

Trp Val Gln Ile Tyr Gly Asp Asn Lys Phe Ile Glu Lys Ala Phe Thr

365 370 375

tat gca aga aaa tat gct ccg gca aat tgt aag ctt tac tac aac gat 1384

Tyr Ala Arg Lys Tyr Ala Pro Ala Asn Cys Lys Leu Tyr Tyr Asn Asp

380 385 390 395

tac aac gaa tat tgg gat cat aag aga gac tgt att gcc tca att tgt 1432

Tyr Asn Glu Tyr Trp Asp His Lys Arg Asp Cys Ile Ala Ser Ile Cys

400 405 410

gca aac ttg tac aac aag ggc ttg ctt gac ggt gtg gga atg cag tcc 1480

Ala Asn Leu Tyr Asn Lys Gly Leu Leu Asp Gly Val Gly Met Gln Ser

415 420 425

cat att aat gcg gat atg aat gga ttc tca ggt ata caa aat tat aaa 1528

His Ile Asn Ala Asp Met Asn Gly Phe Ser Gly Ile Gln Asn Tyr Lys

430 435 440

gca gct ttg cag aaa tat ata aat atc ggt tgt gat gtc caa att acc 1576

Ala Ala Leu Gln Lys Tyr Ile Asn Ile Gly Cys Asp Val Gln Ile Thr

445 450 455

gag ctt gat att agt aca gaa aac ggc aaa ttt agc tta cag cag cag 1624

Glu Leu Asp Ile Ser Thr Glu Asn Gly Lys Phe Ser Leu Gln Gln Gln

460 465 470 475

gct gat aaa tat aaa gct gtt ttc cag gca gct gtt gat ata aac aga 1672

Ala Asp Lys Tyr Lys Ala Val Phe Gln Ala Ala Val Asp Ile Asn Arg

480 485 490

acc tcc agc aaa gga aag gtt acg gct gtc tgt gta tgg gga cct aat 1720

Thr Ser Ser Lys Gly Lys Val Thr Ala Val Cys Val Trp Gly Pro Asn

495 500 505

gac gcc aat act tgg ctc ggt tca caa aat gca cct ctt ttg ttt aac 1768

Asp Ala Asn Thr Trp Leu Gly Ser Gln Asn Ala Pro Leu Leu Phe Asn

510 515 520

gca aac aat caa ccg aaa ccg gca tac aat gcg gtt gca tcc att att 1816

Ala Asn Asn Gln Pro Lys Pro Ala Tyr Asn Ala Val Ala Ser Ile Ile

525 530 535

cct cag tcc gaa tgg ggc gac ggt aac aat ccg gcc ggc ggc gga gga 1864

Pro Gln Ser Glu Trp Gly Asp Gly Asn Asn Pro Ala Gly Gly Gly Gly

540 545 550 555

gga ggc aaa ccg gaa gag ccg gat gca aac gga tat tat tat cat gac 1912

Gly Gly Lys Pro Glu Glu Pro Asp Ala Asn Gly Tyr Tyr Tyr His Asp

560 565 570

act ttt gaa gga agc gta gga cag tgg aca gcc aga gga cct gcg gaa 1960

Thr Phe Glu Gly Ser Val Gly Gln Trp Thr Ala Arg Gly Pro Ala Glu

575 580 585

gtt ctg ctt agc gga aga acg gct tac aaa ggt tca gaa tca ctc ttg 2008

Val Leu Leu Ser Gly Arg Thr Ala Tyr Lys Gly Ser Glu Ser Leu Leu

590 595 600

gta agg aac cgt acg gca gca tgg aac gga gca caa cgg gcg ctg aat 2056

Val Arg Asn Arg Thr Ala Ala Trp Asn Gly Ala Gln Arg Ala Leu Asn

605 610 615

ccc aga acg ttt gtt ccc gga aac aca tat tgt ttc agc gta gtg gca 2104

Pro Arg Thr Phe Val Pro Gly Asn Thr Tyr Cys Phe Ser Val Val Ala

620 625 630 635

tcg ttt att gaa ggt gcg tct tcc aca aca ttc tgc atg aag ctg caa 2152

Ser Phe Ile Glu Gly Ala Ser Ser Thr Thr Phe Cys Met Lys Leu Gln

640 645 650

tac gta gac gga agc ggc act caa cgg tat gat acc ata gat atg aaa 2200

Tyr Val Asp Gly Ser Gly Thr Gln Arg Tyr Asp Thr Ile Asp Met Lys

655 660 665

act gtg ggt cca aat cag tgg gtt cac ctg tac aat ccg caa tac aga 2248

Thr Val Gly Pro Asn Gln Trp Val His Leu Tyr Asn Pro Gln Tyr Arg

670 675 680

att cct tcc gat gca aca gat atg tat gtt tat gtg gaa aca gcg gat 2296

Ile Pro Ser Asp Ala Thr Asp Met Tyr Val Tyr Val Glu Thr Ala Asp

685 690 695

gac acc att aac ttc tac ata gat gag gca atc gga gcg gtt gcc gga 2344

Asp Thr Ile Asn Phe Tyr Ile Asp Glu Ala Ile Gly Ala Val Ala Gly

700 705 710 715

act gta atc gaa gga cct gct cca cag cct aca cag cct ccg gta ctg 2392

Thr Val Ile Glu Gly Pro Ala Pro Gln Pro Thr Gln Pro Pro Val Leu

720 725 730

ctt ggc gat gta aac ggt gat gga acc att aac tca act gac ttg aca 2440

Leu Gly Asp Val Asn Gly Asp Gly Thr Ile Asn Ser Thr Asp Leu Thr

735 740 745

atg tta aag aga agc gtg ttg agg gca atc acc ctt acc gac gat gca 2488

Met Leu Lys Arg Ser Val Leu Arg Ala Ile Thr Leu Thr Asp Asp Ala

750 755 760

aag gct aga gca gac gtt gac aag aat gga tcg ata aac agc act gat 2536

Lys Ala Arg Ala Asp Val Asp Lys Asn Gly Ser Ile Asn Ser Thr Asp

765 770 775

gtt tta ctt ctt tca cgc tac ctt tta aga gta atc gac aaa ttt cct 2584

Val Leu Leu Leu Ser Arg Tyr Leu Leu Arg Val Ile Asp Lys Phe Pro

780 785 790 795

gta gca gaa aat cct tct tct tct ttt aaa tat gag tcg gcc gtg caa 2632

Val Ala Glu Asn Pro Ser Ser Ser Phe Lys Tyr Glu Ser Ala Val Gln

800 805 810

tat cgg ccg gct cct gat tct tat tta aac cct tgt ccg cag gcg gga 2680

Tyr Arg Pro Ala Pro Asp Ser Tyr Leu Asn Pro Cys Pro Gln Ala Gly

815 820 825

aga att gtc aag gaa aca tat aca gga ata aac gga act aag agt ctt 2728

Arg Ile Val Lys Glu Thr Tyr Thr Gly Ile Asn Gly Thr Lys Ser Leu

830 835 840

aat gta tat ctt cca tac ggt tat gat ccg aac aaa aaa tat aac att 2776

Asn Val Tyr Leu Pro Tyr Gly Tyr Asp Pro Asn Lys Lys Tyr Asn Ile

845 850 855

ttc tac ctt atg cat ggc ggc ggt gaa aat gag aat acg att ttc agc 2824

Phe Tyr Leu Met His Gly Gly Gly Glu Asn Glu Asn Thr Ile Phe Ser

860 865 870 875

aac gat gtt aaa ttg caa aat atc ctt gac cac gcg att atg aac ggt 2872

Asn Asp Val Lys Leu Gln Asn Ile Leu Asp His Ala Ile Met Asn Gly

880 885 890

gaa ctt gag cct ttg att gta gta aca ccc act ttc aac ggc gga aac 2920

Glu Leu Glu Pro Leu Ile Val Val Thr Pro Thr Phe Asn Gly Gly Asn

895 900 905

tgc acg gcc caa aac ttt tat cag gaa ttc agg caa aat gtc att cct 2968

Cys Thr Ala Gln Asn Phe Tyr Gln Glu Phe Arg Gln Asn Val Ile Pro

910 915 920

ttt gtg gaa agc aag tac tct act tat gca gaa tca aca acc cca cag 3016

Phe Val Glu Ser Lys Tyr Ser Thr Tyr Ala Glu Ser Thr Thr Pro Gln

925 930 935

gga ata gcc gct tca aga atg cac aga ggt ttc ggc gga ttc tca atg 3064

Gly Ile Ala Ala Ser Arg Met His Arg Gly Phe Gly Gly Phe Ser Met

940 945 950 955

gga gga ttg aca aca tgg tat gta atg gtt aac tgc ctt gat tac gtt 3112

Gly Gly Leu Thr Thr Trp Tyr Val Met Val Asn Cys Leu Asp Tyr Val

960 965 970

gca tat ttt atg cct tta agc ggt gac tac tgg tat gga aac agt ccg 3160

Ala Tyr Phe Met Pro Leu Ser Gly Asp Tyr Trp Tyr Gly Asn Ser Pro

975 980 985

cag gat aag gct aat tca att gct gaa gca att aac aga tcc gga ctt 3208

Gln Asp Lys Ala Asn Ser Ile Ala Glu Ala Ile Asn Arg Ser Gly Leu

990 995 1000

tca aag agg gag tat ttc gta ttt gcg gcc acc ggt tcc gac cat att 3256

Ser Lys Arg Glu Tyr Phe Val Phe Ala Ala Thr Gly Ser Asp His Ile

1005 1010 1015

gca tat gct aat atg aat cct caa att gaa gct atg aag gct ttg ccg 3304

Ala Tyr Ala Asn Met Asn Pro Gln Ile Glu Ala Met Lys Ala Leu Pro

1020 1025 1030 1035

cat ttt gat tat act tcg gat ttt tcc aaa ggt aat ttt tac ttt ctt 3352

His Phe Asp Tyr Thr Ser Asp Phe Ser Lys Gly Asn Phe Tyr Phe Leu

1040 1045 1050

gta gct ccg ggc gcc act cac tgg tgg gga tac gta aga cat tat att 3400

Val Ala Pro Gly Ala Thr His Trp Trp Gly Tyr Val Arg His Tyr Ile

1055 1060 1065

tat gat gca ctt cca tat ttc ttc cat gaa tgaatgagaa agaaaaacat 3450

Tyr Asp Ala Leu Pro Tyr Phe Phe His Glu

1070 1075

gattgagttt gtaatcaata aaaaaaggaa ttttttagtg gtgtccaggt tattgaa 3507

12

1077

Clostridium thermocellum

12

Met Lys Asn Lys Arg Val Leu Ala Lys Ile Thr Ala Leu Val Val Leu

1 5 10 15

Leu Gly Val Phe Phe Val Leu Pro Ser Asn Ile Ser Gln Leu Tyr Ala

20 25 30

Asp Tyr Glu Val Val His Asp Thr Phe Glu Val Asn Phe Asp Gly Trp

35 40 45

Cys Asn Leu Gly Val Asp Thr Tyr Leu Thr Ala Val Glu Asn Glu Gly

50 55 60

Asn Asn Gly Thr Arg Gly Met Met Val Ile Asn Arg Ser Ser Ala Ser

65 70 75 80

Asp Gly Ala Tyr Ser Glu Lys Gly Phe Tyr Leu Asp Gly Gly Val Glu

85 90 95

Tyr Lys Tyr Ser Val Phe Val Lys His Asn Gly Thr Gly Thr Glu Thr

100 105 110

Phe Lys Leu Ser Val Ser Tyr Leu Asp Ser Glu Thr Glu Glu Glu Asn

115 120 125

Lys Glu Val Ile Ala Thr Lys Asp Val Val Ala Gly Glu Trp Thr Glu

130 135 140

Ile Ser Ala Lys Tyr Lys Ala Pro Lys Thr Ala Val Asn Ile Thr Leu

145 150 155 160

Ser Ile Thr Thr Asp Ser Thr Val Asp Phe Ile Phe Asp Asp Val Thr

165 170 175

Ile Thr Arg Lys Gly Met Ala Glu Ala Asn Thr Val Tyr Ala Ala Asn

180 185 190

Ala Val Leu Lys Asp Met Tyr Ala Asn Tyr Phe Arg Val Gly Ser Val

195 200 205

Leu Asn Ser Gly Thr Val Asn Asn Ser Ser Ile Lys Ala Leu Ile Leu

210 215 220

Arg Glu Phe Asn Ser Ile Thr Cys Glu Asn Glu Met Lys Pro Asp Ala

225 230 235 240

Thr Leu Val Gln Ser Gly Ser Thr Asn Thr Asn Ile Arg Val Ser Leu

245 250 255

Asn Arg Ala Ala Ser Ile Leu Asn Phe Cys Ala Gln Asn Asn Ile Ala

260 265 270

Val Arg Gly His Thr Leu Val Trp His Ser Gln Thr Pro Gln Trp Phe

275 280 285

Phe Lys Asp Asn Phe Gln Asp Asn Gly Asn Trp Val Ser Gln Ser Val

290 295 300

Met Asp Gln Arg Leu Glu Ser Tyr Ile Lys Asn Met Phe Ala Glu Ile

305 310 315 320

Gln Arg Gln Tyr Pro Ser Leu Asn Leu Tyr Ala Tyr Asp Val Val Asn

325 330 335

Glu Ala Val Ser Asp Asp Ala Asn Arg Thr Arg Tyr Tyr Gly Gly Ala

340 345 350

Arg Glu Pro Gly Tyr Gly Asn Gly Arg Ser Pro Trp Val Gln Ile Tyr

355 360 365

Gly Asp Asn Lys Phe Ile Glu Lys Ala Phe Thr Tyr Ala Arg Lys Tyr

370 375 380

Ala Pro Ala Asn Cys Lys Leu Tyr Tyr Asn Asp Tyr Asn Glu Tyr Trp

385 390 395 400

Asp His Lys Arg Asp Cys Ile Ala Ser Ile Cys Ala Asn Leu Tyr Asn

405 410 415

Lys Gly Leu Leu Asp Gly Val Gly Met Gln Ser His Ile Asn Ala Asp

420 425 430

Met Asn Gly Phe Ser Gly Ile Gln Asn Tyr Lys Ala Ala Leu Gln Lys

435 440 445

Tyr Ile Asn Ile Gly Cys Asp Val Gln Ile Thr Glu Leu Asp Ile Ser

450 455 460

Thr Glu Asn Gly Lys Phe Ser Leu Gln Gln Gln Ala Asp Lys Tyr Lys

465 470 475 480

Ala Val Phe Gln Ala Ala Val Asp Ile Asn Arg Thr Ser Ser Lys Gly

485 490 495

Lys Val Thr Ala Val Cys Val Trp Gly Pro Asn Asp Ala Asn Thr Trp

500 505 510

Leu Gly Ser Gln Asn Ala Pro Leu Leu Phe Asn Ala Asn Asn Gln Pro

515 520 525

Lys Pro Ala Tyr Asn Ala Val Ala Ser Ile Ile Pro Gln Ser Glu Trp

530 535 540

Gly Asp Gly Asn Asn Pro Ala Gly Gly Gly Gly Gly Gly Lys Pro Glu

545 550 555 560

Glu Pro Asp Ala Asn Gly Tyr Tyr Tyr His Asp Thr Phe Glu Gly Ser

565 570 575

Val Gly Gln Trp Thr Ala Arg Gly Pro Ala Glu Val Leu Leu Ser Gly

580 585 590

Arg Thr Ala Tyr Lys Gly Ser Glu Ser Leu Leu Val Arg Asn Arg Thr

595 600 605

Ala Ala Trp Asn Gly Ala Gln Arg Ala Leu Asn Pro Arg Thr Phe Val

610 615 620

Pro Gly Asn Thr Tyr Cys Phe Ser Val Val Ala Ser Phe Ile Glu Gly

625 630 635 640

Ala Ser Ser Thr Thr Phe Cys Met Lys Leu Gln Tyr Val Asp Gly Ser

645 650 655

Gly Thr Gln Arg Tyr Asp Thr Ile Asp Met Lys Thr Val Gly Pro Asn

660 665 670

Gln Trp Val His Leu Tyr Asn Pro Gln Tyr Arg Ile Pro Ser Asp Ala

675 680 685

Thr Asp Met Tyr Val Tyr Val Glu Thr Ala Asp Asp Thr Ile Asn Phe

690 695 700

Tyr Ile Asp Glu Ala Ile Gly Ala Val Ala Gly Thr Val Ile Glu Gly

705 710 715 720

Pro Ala Pro Gln Pro Thr Gln Pro Pro Val Leu Leu Gly Asp Val Asn

725 730 735

Gly Asp Gly Thr Ile Asn Ser Thr Asp Leu Thr Met Leu Lys Arg Ser

740 745 750

Val Leu Arg Ala Ile Thr Leu Thr Asp Asp Ala Lys Ala Arg Ala Asp

755 760 765

Val Asp Lys Asn Gly Ser Ile Asn Ser Thr Asp Val Leu Leu Leu Ser

770 775 780

Arg Tyr Leu Leu Arg Val Ile Asp Lys Phe Pro Val Ala Glu Asn Pro

785 790 795 800

Ser Ser Ser Phe Lys Tyr Glu Ser Ala Val Gln Tyr Arg Pro Ala Pro

805 810 815

Asp Ser Tyr Leu Asn Pro Cys Pro Gln Ala Gly Arg Ile Val Lys Glu

820 825 830

Thr Tyr Thr Gly Ile Asn Gly Thr Lys Ser Leu Asn Val Tyr Leu Pro

835 840 845

Tyr Gly Tyr Asp Pro Asn Lys Lys Tyr Asn Ile Phe Tyr Leu Met His

850 855 860

Gly Gly Gly Glu Asn Glu Asn Thr Ile Phe Ser Asn Asp Val Lys Leu

865 870 875 880

Gln Asn Ile Leu Asp His Ala Ile Met Asn Gly Glu Leu Glu Pro Leu

885 890 895

Ile Val Val Thr Pro Thr Phe Asn Gly Gly Asn Cys Thr Ala Gln Asn

900 905 910

Phe Tyr Gln Glu Phe Arg Gln Asn Val Ile Pro Phe Val Glu Ser Lys

915 920 925

Tyr Ser Thr Tyr Ala Glu Ser Thr Thr Pro Gln Gly Ile Ala Ala Ser

930 935 940

Arg Met His Arg Gly Phe Gly Gly Phe Ser Met Gly Gly Leu Thr Thr

945 950 955 960

Trp Tyr Val Met Val Asn Cys Leu Asp Tyr Val Ala Tyr Phe Met Pro

965 970 975

Leu Ser Gly Asp Tyr Trp Tyr Gly Asn Ser Pro Gln Asp Lys Ala Asn

980 985 990

Ser Ile Ala Glu Ala Ile Asn Arg Ser Gly Leu Ser Lys Arg Glu Tyr

995 1000 1005

Phe Val Phe Ala Ala Thr Gly Ser Asp His Ile Ala Tyr Ala Asn Met

1010 1015 1020

Asn Pro Gln Ile Glu Ala Met Lys Ala Leu Pro His Phe Asp Tyr Thr

1025 1030 1035 1040

Ser Asp Phe Ser Lys Gly Asn Phe Tyr Phe Leu Val Ala Pro Gly Ala

1045 1050 1055

Thr His Trp Trp Gly Tyr Val Arg His Tyr Ile Tyr Asp Ala Leu Pro

1060 1065 1070

Tyr Phe Phe His Glu

1075

13

atatataaat aagggtatta attctgcaaa aagaaaagtg tttgctacat gaggtccatt 60

aatttttatt ttatatcata aatcaaaaag gaggagaaac atg tca aga aaa ctt 115

Met Ser Arg Lys Leu

1 5

ttc agt gta tta ctt gtt ggc ttg atg ctt atg aca tcg ttg ctt gtc 163

Phe Ser Val Leu Leu Val Gly Leu Met Leu Met Thr Ser Leu Leu Val

10 15 20

aca ata agc agt aca tca gcg gca tcc ttg cca acc atg ccg cct tcg 211

Thr Ile Ser Ser Thr Ser Ala Ala Ser Leu Pro Thr Met Pro Pro Ser

25 30 35

gga tat gac cag gta agg aac ggc gtt ccg aga ggg cag gtc gta aat 259

Gly Tyr Asp Gln Val Arg Asn Gly Val Pro Arg Gly Gln Val Val Asn

40 45 50

att tct tat ttc tcc acg gcc acc aac agt acc agg ccg gca aga gtt 307

Ile Ser Tyr Phe Ser Thr Ala Thr Asn Ser Thr Arg Pro Ala Arg Val

55 60 65

tat ttg ccg ccg gga tat tca aag gac aaa aaa tac agt gtt ttg tat 355

Tyr Leu Pro Pro Gly Tyr Ser Lys Asp Lys Lys Tyr Ser Val Leu Tyr

70 75 80 85

ctc tta cac ggc ata ggc ggt agt gaa aac gac tgg ttc gaa ggg gga 403

Leu Leu His Gly Ile Gly Gly Ser Glu Asn Asp Trp Phe Glu Gly Gly

90 95 100

ggc aga gcc aat gtt att gcc gac aat ctg att gcc gag gga aaa atc 451

Gly Arg Ala Asn Val Ile Ala Asp Asn Leu Ile Ala Glu Gly Lys Ile

105 110 115

aag ccc ctg ata att gta aca ccg aat act aac gcc gcc ggt ccg gga 499

Lys Pro Leu Ile Ile Val Thr Pro Asn Thr Asn Ala Ala Gly Pro Gly

120 125 130

ata gcg gac ggt tat gaa aat ttc aca aaa gat ttg ctc aac agt ctt 547

Ile Ala Asp Gly Tyr Glu Asn Phe Thr Lys Asp Leu Leu Asn Ser Leu

135 140 145

att ccc tat atc gaa tct aac tat tca gtc tac acc gac cgc gaa cat 595

Ile Pro Tyr Ile Glu Ser Asn Tyr Ser Val Tyr Thr Asp Arg Glu His

150 155 160 165

cgg gcg att gca gga ctt tca atg ggt gga gga caa tcg ttt aat att 643

Arg Ala Ile Ala Gly Leu Ser Met Gly Gly Gly Gln Ser Phe Asn Ile

170 175 180

gga ttg acc aat ctc gat aaa ttt gcc tat att ggc ccg att tca gcg 691

Gly Leu Thr Asn Leu Asp Lys Phe Ala Tyr Ile Gly Pro Ile Ser Ala

185 190 195

gct cca aac act tat cca aat gag agg ctt ttt cct gac gga gga aaa 739

Ala Pro Asn Thr Tyr Pro Asn Glu Arg Leu Phe Pro Asp Gly Gly Lys

200 205 210

gct gca agg gag aaa ttg aaa ctg ctc ttt att gcc tgc gga acc aat 787

Ala Ala Arg Glu Lys Leu Lys Leu Leu Phe Ile Ala Cys Gly Thr Asn

215 220 225

gac agt ctg ata ggt ttt gga cag aga gta cat gaa tat tgc gtt gcc 835

Asp Ser Leu Ile Gly Phe Gly Gln Arg Val His Glu Tyr Cys Val Ala

230 235 240 245

aac aac att aac cat gtc tat tgg ctt att cag ggc gga gga cac gat 883

Asn Asn Ile Asn His Val Tyr Trp Leu Ile Gln Gly Gly Gly His Asp

250 255 260

ttt aat gtg tgg aag ccc gga ttg tgg aat ttc ctt caa atg gca gat 931

Phe Asn Val Trp Lys Pro Gly Leu Trp Asn Phe Leu Gln Met Ala Asp

265 270 275

gaa gcc gga ttg acg agg gat gga aac act ccg gtt ccg aca ccc agt 979

Glu Ala Gly Leu Thr Arg Asp Gly Asn Thr Pro Val Pro Thr Pro Ser

280 285 290

cca aag ccg gct aac aca cgt att gaa gcg gaa gat tat gac ggt att 1027

Pro Lys Pro Ala Asn Thr Arg Ile Glu Ala Glu Asp Tyr Asp Gly Ile

295 300 305

aat tct tca agt att gag ata ata ggt gtt cca cct gaa gga ggc aga 1075

Asn Ser Ser Ser Ile Glu Ile Ile Gly Val Pro Pro Glu Gly Gly Arg

310 315 320 325

gga ata ggt tat att acc agt ggt gat tat ctg gta tac aag agt ata 1123

Gly Ile Gly Tyr Ile Thr Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile

330 335 340

gac ttt gga aac gga gca acg tcg ttt aag gcc aag gtt gca aat gca 1171

Asp Phe Gly Asn Gly Ala Thr Ser Phe Lys Ala Lys Val Ala Asn Ala

345 350 355

aat act tcc aat att gaa ctt aga tta aac ggt ccg aat ggt act ctc 1219

Asn Thr Ser Asn Ile Glu Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu

360 365 370

ata ggc aca ctc tcg gta aaa tcc aca gga gat tgg aat aca tat gag 1267

Ile Gly Thr Leu Ser Val Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu

375 380 385

gag caa act tgc agc att agc aaa gtc acc gga ata aat gat ttg tac 1315

Glu Gln Thr Cys Ser Ile Ser Lys Val Thr Gly Ile Asn Asp Leu Tyr

390 395 400 405

ttg gta ttc aaa ggc cct gta aac ata gac tgg ttc act ttt ggc gtt 1363

Leu Val Phe Lys Gly Pro Val Asn Ile Asp Trp Phe Thr Phe Gly Val

410 415 420

gaa agc agt tcc aca ggt ctg ggg gat tta aat ggt gac gga aat att 1411

Glu Ser Ser Ser Thr Gly Leu Gly Asp Leu Asn Gly Asp Gly Asn Ile

425 430 435

aac tcg tcg gac ctt cag gcg tta aag agg cat ttg ctc ggt ata tca 1459

Asn Ser Ser Asp Leu Gln Ala Leu Lys Arg His Leu Leu Gly Ile Ser

440 445 450

ccg ctt acg gga gag gct ctt tta aga gcg gat gta aat agg agc ggc 1507

Pro Leu Thr Gly Glu Ala Leu Leu Arg Ala Asp Val Asn Arg Ser Gly

455 460 465

aaa gtg gat tct act gac tat tca gtg ctg aaa aga tat ata ctc cgc 1555

Lys Val Asp Ser Thr Asp Tyr Ser Val Leu Lys Arg Tyr Ile Leu Arg

470 475 480 485

att att aca gag ttc ccc gga caa ggt gat gta cag aca ccc aat ccg 1603

Ile Ile Thr Glu Phe Pro Gly Gln Gly Asp Val Gln Thr Pro Asn Pro

490 495 500

tct gtt act ccg aca caa act cct atc ccc acg att tcg gga aat gct 1651

Ser Val Thr Pro Thr Gln Thr Pro Ile Pro Thr Ile Ser Gly Asn Ala

505 510 515

ctt agg gat tat gcg gag gca agg gga ata aaa atc gga aca tgt gtc 1699

Leu Arg Asp Tyr Ala Glu Ala Arg Gly Ile Lys Ile Gly Thr Cys Val

520 525 530

aac tat ccg ttt tac aac aat tca gat cca acc tac aac agc att ttg 1747

Asn Tyr Pro Phe Tyr Asn Asn Ser Asp Pro Thr Tyr Asn Ser Ile Leu

535 540 545

caa aga gaa ttt tca atg gtt gta tgt gaa aat gaa atg aag ttt gat 1795

Gln Arg Glu Phe Ser Met Val Val Cys Glu Asn Glu Met Lys Phe Asp

550 555 560 565

gct ttg cag ccg aga caa aac gtt ttt gat ttt tcg aaa gga gac cag 1843

Ala Leu Gln Pro Arg Gln Asn Val Phe Asp Phe Ser Lys Gly Asp Gln

570 575 580

ttg ctt gct ttt gca gaa aga aac ggt atg cag atg agg gga cat acg 1891

Leu Leu Ala Phe Ala Glu Arg Asn Gly Met Gln Met Arg Gly His Thr

585 590 595

ttg att tgg cac aat caa aac ccg tca tgg ctt aca aac ggt aac tgg 1939

Leu Ile Trp His Asn Gln Asn Pro Ser Trp Leu Thr Asn Gly Asn Trp

600 605 610

aac cgg gat tcg ctg ctt gcg gta atg aaa aat cac att acc act gtt 1987

Asn Arg Asp Ser Leu Leu Ala Val Met Lys Asn His Ile Thr Thr Val

615 620 625

atg acc cat tac aaa ggt aaa att gtt gag tgg gat gtg gca aac gaa 2035

Met Thr His Tyr Lys Gly Lys Ile Val Glu Trp Asp Val Ala Asn Glu

630 635 640 645

tgt atg gat gat tcc ggc aac ggc tta aga agc agc ata tgg aga aat 2083

Cys Met Asp Asp Ser Gly Asn Gly Leu Arg Ser Ser Ile Trp Arg Asn

650 655 660

gta atc ggt cag gac tac ctt gac tat gct ttc agg tat gca aga gaa 2131

Val Ile Gly Gln Asp Tyr Leu Asp Tyr Ala Phe Arg Tyr Ala Arg Glu

665 670 675

gca gat ccc gat gca ctt ctt ttc tac aat gat tat aat att gaa gac 2179

Ala Asp Pro Asp Ala Leu Leu Phe Tyr Asn Asp Tyr Asn Ile Glu Asp

680 685 690

ttg ggt cca aag tcc aat gcg gta ttt aac atg att aaa agt atg aag 2227

Leu Gly Pro Lys Ser Asn Ala Val Phe Asn Met Ile Lys Ser Met Lys

695 700 705

gaa aga ggt gtg ccg att gac gga gta gga ttc caa tgc cac ttt atc 2275

Glu Arg Gly Val Pro Ile Asp Gly Val Gly Phe Gln Cys His Phe Ile

710 715 720 725

aat gga atg agc ccc gag tac ctt gcc agc att gat caa aat att aag 2323

Asn Gly Met Ser Pro Glu Tyr Leu Ala Ser Ile Asp Gln Asn Ile Lys

730 735 740

aga tat gcg gaa ata ggc gtt ata gta tcc ttt acc gaa ata gat ata 2371

Arg Tyr Ala Glu Ile Gly Val Ile Val Ser Phe Thr Glu Ile Asp Ile

745 750 755

cgc ata cct cag tcg gaa aac ccg gca act gca ttc cag gta cag gca 2419

Arg Ile Pro Gln Ser Glu Asn Pro Ala Thr Ala Phe Gln Val Gln Ala

760 765 770

aac aac tat aag gaa ctt atg aaa att tgt ctg gca aac ccc aat tgc 2467

Asn Asn Tyr Lys Glu Leu Met Lys Ile Cys Leu Ala Asn Pro Asn Cys

775 780 785

aat acc ttt gta atg tgg gga ttc aca gat aaa tac aca tgg att ccg 2515

Asn Thr Phe Val Met Trp Gly Phe Thr Asp Lys Tyr Thr Trp Ile Pro

790 795 800 805

gga act ttc cca gga tat ggc aat cca ttg att tat gac agc aat tac 2563

Gly Thr Phe Pro Gly Tyr Gly Asn Pro Leu Ile Tyr Asp Ser Asn Tyr

810 815 820

aat ccg aaa ccg gca tac aat gca ata aag gaa gct ctt atg ggc tat 2611

Asn Pro Lys Pro Ala Tyr Asn Ala Ile Lys Glu Ala Leu Met Gly Tyr

825 830 835

tgataattcc gaaaagctga gcagataatg atgccgtaaa gccggcttct gaattaagag 2671

ccggctttac ggagatatac tttttacggc agaatacctg ttatttccat g 2722

14

837

Clostridium thermocellum

14

Met Ser Arg Lys Leu Phe Ser Val Leu Leu Val Gly Leu Met Leu Met

1 5 10 15

Thr Ser Leu Leu Val Thr Ile Ser Ser Thr Ser Ala Ala Ser Leu Pro

20 25 30

Thr Met Pro Pro Ser Gly Tyr Asp Gln Val Arg Asn Gly Val Pro Arg

35 40 45

Gly Gln Val Val Asn Ile Ser Tyr Phe Ser Thr Ala Thr Asn Ser Thr

50 55 60

Arg Pro Ala Arg Val Tyr Leu Pro Pro Gly Tyr Ser Lys Asp Lys Lys

65 70 75 80

Tyr Ser Val Leu Tyr Leu Leu His Gly Ile Gly Gly Ser Glu Asn Asp

85 90 95

Trp Phe Glu Gly Gly Gly Arg Ala Asn Val Ile Ala Asp Asn Leu Ile

100 105 110

Ala Glu Gly Lys Ile Lys Pro Leu Ile Ile Val Thr Pro Asn Thr Asn

115 120 125

Ala Ala Gly Pro Gly Ile Ala Asp Gly Tyr Glu Asn Phe Thr Lys Asp

130 135 140

Leu Leu Asn Ser Leu Ile Pro Tyr Ile Glu Ser Asn Tyr Ser Val Tyr

145 150 155 160

Thr Asp Arg Glu His Arg Ala Ile Ala Gly Leu Ser Met Gly Gly Gly

165 170 175

Gln Ser Phe Asn Ile Gly Leu Thr Asn Leu Asp Lys Phe Ala Tyr Ile

180 185 190

Gly Pro Ile Ser Ala Ala Pro Asn Thr Tyr Pro Asn Glu Arg Leu Phe

195 200 205

Pro Asp Gly Gly Lys Ala Ala Arg Glu Lys Leu Lys Leu Leu Phe Ile

210 215 220

Ala Cys Gly Thr Asn Asp Ser Leu Ile Gly Phe Gly Gln Arg Val His

225 230 235 240

Glu Tyr Cys Val Ala Asn Asn Ile Asn His Val Tyr Trp Leu Ile Gln

245 250 255

Gly Gly Gly His Asp Phe Asn Val Trp Lys Pro Gly Leu Trp Asn Phe

260 265 270

Leu Gln Met Ala Asp Glu Ala Gly Leu Thr Arg Asp Gly Asn Thr Pro

275 280 285

Val Pro Thr Pro Ser Pro Lys Pro Ala Asn Thr Arg Ile Glu Ala Glu

290 295 300

Asp Tyr Asp Gly Ile Asn Ser Ser Ser Ile Glu Ile Ile Gly Val Pro

305 310 315 320

Pro Glu Gly Gly Arg Gly Ile Gly Tyr Ile Thr Ser Gly Asp Tyr Leu

325 330 335

Val Tyr Lys Ser Ile Asp Phe Gly Asn Gly Ala Thr Ser Phe Lys Ala

340 345 350

Lys Val Ala Asn Ala Asn Thr Ser Asn Ile Glu Leu Arg Leu Asn Gly

355 360 365

Pro Asn Gly Thr Leu Ile Gly Thr Leu Ser Val Lys Ser Thr Gly Asp

370 375 380

Trp Asn Thr Tyr Glu Glu Gln Thr Cys Ser Ile Ser Lys Val Thr Gly

385 390 395 400

Ile Asn Asp Leu Tyr Leu Val Phe Lys Gly Pro Val Asn Ile Asp Trp

405 410 415

Phe Thr Phe Gly Val Glu Ser Ser Ser Thr Gly Leu Gly Asp Leu Asn

420 425 430

Gly Asp Gly Asn Ile Asn Ser Ser Asp Leu Gln Ala Leu Lys Arg His

435 440 445

Leu Leu Gly Ile Ser Pro Leu Thr Gly Glu Ala Leu Leu Arg Ala Asp

450 455 460

Val Asn Arg Ser Gly Lys Val Asp Ser Thr Asp Tyr Ser Val Leu Lys

465 470 475 480

Arg Tyr Ile Leu Arg Ile Ile Thr Glu Phe Pro Gly Gln Gly Asp Val

485 490 495

Gln Thr Pro Asn Pro Ser Val Thr Pro Thr Gln Thr Pro Ile Pro Thr

500 505 510

Ile Ser Gly Asn Ala Leu Arg Asp Tyr Ala Glu Ala Arg Gly Ile Lys

515 520 525

Ile Gly Thr Cys Val Asn Tyr Pro Phe Tyr Asn Asn Ser Asp Pro Thr

530 535 540

Tyr Asn Ser Ile Leu Gln Arg Glu Phe Ser Met Val Val Cys Glu Asn

545 550 555 560

Glu Met Lys Phe Asp Ala Leu Gln Pro Arg Gln Asn Val Phe Asp Phe

565 570 575

Ser Lys Gly Asp Gln Leu Leu Ala Phe Ala Glu Arg Asn Gly Met Gln

580 585 590

Met Arg Gly His Thr Leu Ile Trp His Asn Gln Asn Pro Ser Trp Leu

595 600 605

Thr Asn Gly Asn Trp Asn Arg Asp Ser Leu Leu Ala Val Met Lys Asn

610 615 620

His Ile Thr Thr Val Met Thr His Tyr Lys Gly Lys Ile Val Glu Trp

625 630 635 640

Asp Val Ala Asn Glu Cys Met Asp Asp Ser Gly Asn Gly Leu Arg Ser

645 650 655

Ser Ile Trp Arg Asn Val Ile Gly Gln Asp Tyr Leu Asp Tyr Ala Phe

660 665 670

Arg Tyr Ala Arg Glu Ala Asp Pro Asp Ala Leu Leu Phe Tyr Asn Asp

675 680 685

Tyr Asn Ile Glu Asp Leu Gly Pro Lys Ser Asn Ala Val Phe Asn Met

690 695 700

Ile Lys Ser Met Lys Glu Arg Gly Val Pro Ile Asp Gly Val Gly Phe

705 710 715 720

Gln Cys His Phe Ile Asn Gly Met Ser Pro Glu Tyr Leu Ala Ser Ile

725 730 735

Asp Gln Asn Ile Lys Arg Tyr Ala Glu Ile Gly Val Ile Val Ser Phe

740 745 750

Thr Glu Ile Asp Ile Arg Ile Pro Gln Ser Glu Asn Pro Ala Thr Ala

755 760 765

Phe Gln Val Gln Ala Asn Asn Tyr Lys Glu Leu Met Lys Ile Cys Leu

770 775 780

Ala Asn Pro Asn Cys Asn Thr Phe Val Met Trp Gly Phe Thr Asp Lys

785 790 795 800

Tyr Thr Trp Ile Pro Gly Thr Phe Pro Gly Tyr Gly Asn Pro Leu Ile

805 810 815

Tyr Asp Ser Asn Tyr Asn Pro Lys Pro Ala Tyr Asn Ala Ile Lys Glu

820 825 830

Ala Leu Met Gly Tyr

835

15

gatctttttc ataagtatgc ccccattatt aagtttttta gatgcttgcc tataatttcc 60

cttctggttt tgtgaacttc ttaacggtca gagttcacac tttctttata tattgtctat 120

attataatgt atattgtagt aataatatac caaaattttc ctttaagtaa caatatcttt 180

accctattta gcaattttta acgatatttt ataatttgat tatttttaaa ctatacagtg 240

taaatactat tatttaaaaa gtccaccaaa aatgtaaaat acaatgatat cttaaacgta 300

aaaacctgta caatgattgt tcatcttttt acattattgt tatatatcgt cttggtatag 360

tcagcaattt ttagtcaaga tatacaaggt ccgcaaattt taacttgcaa ttaacaggtc 420

agatgtttta taatgatatc atagaaataa aaggagcact tggctcctta tggggattac 480

tgaaatcata agtttgcttt ttttctaaaa aacaaaggag tgattgaa gtg aaa aaa 537

Val Lys Lys

1

aca gtt aaa caa ttc atc agc agt gcc gtt aca gcg tta atg gtg gct 585

Thr Val Lys Gln Phe Ile Ser Ser Ala Val Thr Ala Leu Met Val Ala

5 10 15

gca agc ctg cct gcc gtt cct tcc gtg aac gca gcc gac gcc cag cag 633

Ala Ser Leu Pro Ala Val Pro Ser Val Asn Ala Ala Asp Ala Gln Gln

20 25 30 35

aga ggc aat atc ggc ggt ttc gat tac gaa atg tgg aac cag aac ggt 681

Arg Gly Asn Ile Gly Gly Phe Asp Tyr Glu Met Trp Asn Gln Asn Gly

40 45 50

cag gga cag gta tca atg acg cct aag gca ggc tct ttc acc tgc tca 729

Gln Gly Gln Val Ser Met Thr Pro Lys Ala Gly Ser Phe Thr Cys Ser

55 60 65

tgg agc aac att gaa aac ttc ctc gca cgt atg ggc aag aac tac gac 777

Trp Ser Asn Ile Glu Asn Phe Leu Ala Arg Met Gly Lys Asn Tyr Asp

70 75 80

agc cag aaa aag aac tac aag gct ttc gga gac att acc ctc tcc tac 825

Ser Gln Lys Lys Asn Tyr Lys Ala Phe Gly Asp Ile Thr Leu Ser Tyr

85 90 95

gac gta gag tac acc ccc aag ggc aac tct tat atg tgc gta tac ggc 873

Asp Val Glu Tyr Thr Pro Lys Gly Asn Ser Tyr Met Cys Val Tyr Gly

100 105 110 115

tgg acg agg aac cct ctc atg gaa tac tac atc gtc gaa ggc tgg ggc 921

Trp Thr Arg Asn Pro Leu Met Glu Tyr Tyr Ile Val Glu Gly Trp Gly

120 125 130

gac tgg cgt cca ccc gga aat gac ggc gaa aac aag ggt aca gtt acc 969

Asp Trp Arg Pro Pro Gly Asn Asp Gly Glu Asn Lys Gly Thr Val Thr

135 140 145

ctg aac ggc aac acc tac gat atc cgc aaa aca atg cgt tat aat cag 1017

Leu Asn Gly Asn Thr Tyr Asp Ile Arg Lys Thr Met Arg Tyr Asn Gln

150 155 160

cca tct ctg gac ggc acg gct aca ttc cct cag tac tgg agc gta cgt 1065

Pro Ser Leu Asp Gly Thr Ala Thr Phe Pro Gln Tyr Trp Ser Val Arg

165 170 175

cag aag agc ggt tca cag aat aat acc acc aac tat atg aag ggt act 1113

Gln Lys Ser Gly Ser Gln Asn Asn Thr Thr Asn Tyr Met Lys Gly Thr

180 185 190 195

atc agc gta tcc aag cac ttt gac gca tgg tca aag gca ggt ctg gat 1161

Ile Ser Val Ser Lys His Phe Asp Ala Trp Ser Lys Ala Gly Leu Asp

200 205 210

atg agc ggt act ctc tac gag gta tcc ctc aac atc gag ggc tac aga 1209

Met Ser Gly Thr Leu Tyr Glu Val Ser Leu Asn Ile Glu Gly Tyr Arg

215 220 225

tca agc gga aac gct aac gtt aaa gct atc tca ttc gac ggc agt ata 1257

Ser Ser Gly Asn Ala Asn Val Lys Ala Ile Ser Phe Asp Gly Ser Ile

230 235 240

ccc gag ccc aca agc gag ccc gta act cag ccc gtt gtc aag gca gag 1305

Pro Glu Pro Thr Ser Glu Pro Val Thr Gln Pro Val Val Lys Ala Glu

245 250 255

cct gac gca aac ggc tac tac ttc aaa gaa aaa ttc gag agc ggc gca 1353

Pro Asp Ala Asn Gly Tyr Tyr Phe Lys Glu Lys Phe Glu Ser Gly Ala

260 265 270 275

ggc gac tgg tca gcc cgc gga aca gga gct aag gta aca agc tct gac 1401

Gly Asp Trp Ser Ala Arg Gly Thr Gly Ala Lys Val Thr Ser Ser Asp

280 285 290

gga ttc aac ggt tca aag ggc ata ctg gta tca gga cgc ggc gac aac 1449

Gly Phe Asn Gly Ser Lys Gly Ile Leu Val Ser Gly Arg Gly Asp Asn

295 300 305

tgg cac ggc gca cag ctc aca ctc gac tca agt gct ttc aca gca ggc 1497

Trp His Gly Ala Gln Leu Thr Leu Asp Ser Ser Ala Phe Thr Ala Gly

310 315 320

gaa aca tac agc ttc ggc gca ctt gta aag cag gac ggc gag tcc tca 1545

Glu Thr Tyr Ser Phe Gly Ala Leu Val Lys Gln Asp Gly Glu Ser Ser

325 330 335

aca gct atg aag ctc act ctc cag tat aac gac gca agc ggc aca gcc 1593

Thr Ala Met Lys Leu Thr Leu Gln Tyr Asn Asp Ala Ser Gly Thr Ala

340 345 350 355

aat tac gat aag gtg gca gag ttc aca gct cca aag ggt gaa tgg gta 1641

Asn Tyr Asp Lys Val Ala Glu Phe Thr Ala Pro Lys Gly Glu Trp Val

360 365 370

gac ctt tcc aat aca tcg ttc act atc ccg tca ggc gct tca gac ctc 1689

Asp Leu Ser Asn Thr Ser Phe Thr Ile Pro Ser Gly Ala Ser Asp Leu

375 380 385

att ctc tat gtt gaa gct ccc gac agc ctt acg gat ttc tat atc gac 1737

Ile Leu Tyr Val Glu Ala Pro Asp Ser Leu Thr Asp Phe Tyr Ile Asp

390 395 400

aac gct ttc ggc ggc atc aag aac aca tct cct ctt gaa gat gtc gga 1785

Asn Ala Phe Gly Gly Ile Lys Asn Thr Ser Pro Leu Glu Asp Val Gly

405 410 415

agc cat act atc agc act ccg ggc agc gag aca aca aca gtc aca act 1833

Ser His Thr Ile Ser Thr Pro Gly Ser Glu Thr Thr Thr Val Thr Thr

420 425 430 435

gca tca aat aag ggt atc aga ggc gat atc aac ggc gac ggc gtt atc 1881

Ala Ser Asn Lys Gly Ile Arg Gly Asp Ile Asn Gly Asp Gly Val Ile

440 445 450

aac tca ttc gac ctt gct cct ctc aga aga ggc att ctc aag atg atg 1929

Asn Ser Phe Asp Leu Ala Pro Leu Arg Arg Gly Ile Leu Lys Met Met

455 460 465

tca ggc agc ggc tcg act ccc gaa aat gct gac gta aac ggc gac ggc 1977

Ser Gly Ser Gly Ser Thr Pro Glu Asn Ala Asp Val Asn Gly Asp Gly

470 475 480

act gta aat gtt gca gac ctc ctg ctt ctc cag aag ttt ata ctc ggt 2025

Thr Val Asn Val Ala Asp Leu Leu Leu Leu Gln Lys Phe Ile Leu Gly

485 490 495

atg gag aag tca ttc ccc gat cct gta aca act acc acg acc aag ccg 2073

Met Glu Lys Ser Phe Pro Asp Pro Val Thr Thr Thr Thr Thr Lys Pro

500 505 510 515

ata aca aca act acc gag aag ata gtt acc aca act act tct tca tct 2121

Ile Thr Thr Thr Thr Glu Lys Ile Val Thr Thr Thr Thr Ser Ser Ser

520 525 530

tct tca agc tca ggc aag aac ctc aat gca gat atc cgc aag gat atg 2169

Ser Ser Ser Ser Gly Lys Asn Leu Asn Ala Asp Ile Arg Lys Asp Met

535 540 545

cct act tca gtt ccc ggc gga aac gaa aag agc ggc ggc tgc aag gtc 2217

Pro Thr Ser Val Pro Gly Gly Asn Glu Lys Ser Gly Gly Cys Lys Val

550 555 560

gag aag aag aca tac aac tgc aag ttc aca ggc ggt cag aag agc tgc 2265

Glu Lys Lys Thr Tyr Asn Cys Lys Phe Thr Gly Gly Gln Lys Ser Cys

565 570 575

aac gtt atc ctg cct cct aac tac agc gca agc aag cag tac cct gtt 2313

Asn Val Ile Leu Pro Pro Asn Tyr Ser Ala Ser Lys Gln Tyr Pro Val

580 585 590 595

atg tac gtt ctc cac ggt atc ggc gga aac gag gga agc atg gta agc 2361

Met Tyr Val Leu His Gly Ile Gly Gly Asn Glu Gly Ser Met Val Ser

600 605 610

ggc atg ggc gtt cag gag ctt ctt gca gga ctt acc gca aac ggc aag 2409

Gly Met Gly Val Gln Glu Leu Leu Ala Gly Leu Thr Ala Asn Gly Lys

615 620 625

gca gag gaa atg ata atc gtt ctc ccg agc cag tac acc agc aag aac 2457

Ala Glu Glu Met Ile Ile Val Leu Pro Ser Gln Tyr Thr Ser Lys Asn

630 635 640

ggc aat cag ggc ggc ggc ttc gga atc aat cag gaa gta tgc gca gct 2505

Gly Asn Gln Gly Gly Gly Phe Gly Ile Asn Gln Glu Val Cys Ala Ala

645 650 655

tac gat aac ttc ctc tat gat atc tca gac agc ctt atc cca ttc atc 2553

Tyr Asp Asn Phe Leu Tyr Asp Ile Ser Asp Ser Leu Ile Pro Phe Ile

660 665 670 675

gag gct aac tat ccc gtt aag aca ggc aga gaa aac cgt gct atc aca 2601

Glu Ala Asn Tyr Pro Val Lys Thr Gly Arg Glu Asn Arg Ala Ile Thr

680 685 690

ggc ttc tca atg ggc gga cgt gaa gct atc tat atc ggt ctt atg cgt 2649

Gly Phe Ser Met Gly Gly Arg Glu Ala Ile Tyr Ile Gly Leu Met Arg

695 700 705

ccc gac ctc ttc gct tac gtt ggc gga gct tgc cct gca ccc ggt atc 2697

Pro Asp Leu Phe Ala Tyr Val Gly Gly Ala Cys Pro Ala Pro Gly Ile

710 715 720

acc cca ggc aag gat atg ttc atg gag cac cca ggc tgt atg cag gag 2745

Thr Pro Gly Lys Asp Met Phe Met Glu His Pro Gly Cys Met Gln Glu

725 730 735

agc gaa atg aag ttc aga gac gtt gga cct gag ccg aat gta ttc atg 2793

Ser Glu Met Lys Phe Arg Asp Val Gly Pro Glu Pro Asn Val Phe Met

740 745 750 755

ata aca ggc ggc aca aac gac ggc gtc gta gga aca ttc ccc aag cag 2841

Ile Thr Gly Gly Thr Asn Asp Gly Val Val Gly Thr Phe Pro Lys Gln

760 765 770

tac agc gat atc ctt aca aga aac ggc gtt gac caa cgt tta cca gtc 2889

Tyr Ser Asp Ile Leu Thr Arg Asn Gly Val Asp Gln Arg Leu Pro Val

775 780 785

tat ccc taacggcgga cacgacgcag gctctgtaaa gcctcatctc tacacattca 2945

Tyr Pro

tgagatacgc attcaaataa tgatatagtt gacatatgaa ggacagcgct ttatgcgctg 3005

tctttctttt tgtgcaaaaa gaaaagccat ttgagctttt gaagctcaaa tggcttatat 3065

ttataatagt atagcttatt ctgttctgag agcctccaca 3105

16

789

Ruminococcus sp.

16

Val Lys Lys Thr Val Lys Gln Phe Ile Ser Ser Ala Val Thr Ala Leu

1 5 10 15

Met Val Ala Ala Ser Leu Pro Ala Val Pro Ser Val Asn Ala Ala Asp

20 25 30

Ala Gln Gln Arg Gly Asn Ile Gly Gly Phe Asp Tyr Glu Met Trp Asn

35 40 45

Gln Asn Gly Gln Gly Gln Val Ser Met Thr Pro Lys Ala Gly Ser Phe

50 55 60

Thr Cys Ser Trp Ser Asn Ile Glu Asn Phe Leu Ala Arg Met Gly Lys

65 70 75 80

Asn Tyr Asp Ser Gln Lys Lys Asn Tyr Lys Ala Phe Gly Asp Ile Thr

85 90 95

Leu Ser Tyr Asp Val Glu Tyr Thr Pro Lys Gly Asn Ser Tyr Met Cys

100 105 110

Val Tyr Gly Trp Thr Arg Asn Pro Leu Met Glu Tyr Tyr Ile Val Glu

115 120 125

Gly Trp Gly Asp Trp Arg Pro Pro Gly Asn Asp Gly Glu Asn Lys Gly

130 135 140

Thr Val Thr Leu Asn Gly Asn Thr Tyr Asp Ile Arg Lys Thr Met Arg

145 150 155 160

Tyr Asn Gln Pro Ser Leu Asp Gly Thr Ala Thr Phe Pro Gln Tyr Trp

165 170 175

Ser Val Arg Gln Lys Ser Gly Ser Gln Asn Asn Thr Thr Asn Tyr Met

180 185 190

Lys Gly Thr Ile Ser Val Ser Lys His Phe Asp Ala Trp Ser Lys Ala

195 200 205

Gly Leu Asp Met Ser Gly Thr Leu Tyr Glu Val Ser Leu Asn Ile Glu

210 215 220

Gly Tyr Arg Ser Ser Gly Asn Ala Asn Val Lys Ala Ile Ser Phe Asp

225 230 235 240

Gly Ser Ile Pro Glu Pro Thr Ser Glu Pro Val Thr Gln Pro Val Val

245 250 255

Lys Ala Glu Pro Asp Ala Asn Gly Tyr Tyr Phe Lys Glu Lys Phe Glu

260 265 270

Ser Gly Ala Gly Asp Trp Ser Ala Arg Gly Thr Gly Ala Lys Val Thr

275 280 285

Ser Ser Asp Gly Phe Asn Gly Ser Lys Gly Ile Leu Val Ser Gly Arg

290 295 300

Gly Asp Asn Trp His Gly Ala Gln Leu Thr Leu Asp Ser Ser Ala Phe

305 310 315 320

Thr Ala Gly Glu Thr Tyr Ser Phe Gly Ala Leu Val Lys Gln Asp Gly

325 330 335

Glu Ser Ser Thr Ala Met Lys Leu Thr Leu Gln Tyr Asn Asp Ala Ser

340 345 350

Gly Thr Ala Asn Tyr Asp Lys Val Ala Glu Phe Thr Ala Pro Lys Gly

355 360 365

Glu Trp Val Asp Leu Ser Asn Thr Ser Phe Thr Ile Pro Ser Gly Ala

370 375 380

Ser Asp Leu Ile Leu Tyr Val Glu Ala Pro Asp Ser Leu Thr Asp Phe

385 390 395 400

Tyr Ile Asp Asn Ala Phe Gly Gly Ile Lys Asn Thr Ser Pro Leu Glu

405 410 415

Asp Val Gly Ser His Thr Ile Ser Thr Pro Gly Ser Glu Thr Thr Thr

420 425 430

Val Thr Thr Ala Ser Asn Lys Gly Ile Arg Gly Asp Ile Asn Gly Asp

435 440 445

Gly Val Ile Asn Ser Phe Asp Leu Ala Pro Leu Arg Arg Gly Ile Leu

450 455 460

Lys Met Met Ser Gly Ser Gly Ser Thr Pro Glu Asn Ala Asp Val Asn

465 470 475 480

Gly Asp Gly Thr Val Asn Val Ala Asp Leu Leu Leu Leu Gln Lys Phe

485 490 495

Ile Leu Gly Met Glu Lys Ser Phe Pro Asp Pro Val Thr Thr Thr Thr

500 505 510

Thr Lys Pro Ile Thr Thr Thr Thr Glu Lys Ile Val Thr Thr Thr Thr

515 520 525

Ser Ser Ser Ser Ser Ser Ser Gly Lys Asn Leu Asn Ala Asp Ile Arg

530 535 540

Lys Asp Met Pro Thr Ser Val Pro Gly Gly Asn Glu Lys Ser Gly Gly

545 550 555 560

Cys Lys Val Glu Lys Lys Thr Tyr Asn Cys Lys Phe Thr Gly Gly Gln

565 570 575

Lys Ser Cys Asn Val Ile Leu Pro Pro Asn Tyr Ser Ala Ser Lys Gln

580 585 590

Tyr Pro Val Met Tyr Val Leu His Gly Ile Gly Gly Asn Glu Gly Ser

595 600 605

Met Val Ser Gly Met Gly Val Gln Glu Leu Leu Ala Gly Leu Thr Ala

610 615 620

Asn Gly Lys Ala Glu Glu Met Ile Ile Val Leu Pro Ser Gln Tyr Thr

625 630 635 640

Ser Lys Asn Gly Asn Gln Gly Gly Gly Phe Gly Ile Asn Gln Glu Val

645 650 655

Cys Ala Ala Tyr Asp Asn Phe Leu Tyr Asp Ile Ser Asp Ser Leu Ile

660 665 670

Pro Phe Ile Glu Ala Asn Tyr Pro Val Lys Thr Gly Arg Glu Asn Arg

675 680 685

Ala Ile Thr Gly Phe Ser Met Gly Gly Arg Glu Ala Ile Tyr Ile Gly

690 695 700

Leu Met Arg Pro Asp Leu Phe Ala Tyr Val Gly Gly Ala Cys Pro Ala

705 710 715 720

Pro Gly Ile Thr Pro Gly Lys Asp Met Phe Met Glu His Pro Gly Cys

725 730 735

Met Gln Glu Ser Glu Met Lys Phe Arg Asp Val Gly Pro Glu Pro Asn

740 745 750

Val Phe Met Ile Thr Gly Gly Thr Asn Asp Gly Val Val Gly Thr Phe

755 760 765

Pro Lys Gln Tyr Ser Asp Ile Leu Thr Arg Asn Gly Val Asp Gln Arg

770 775 780

Leu Pro Val Tyr Pro

785

17

gtt gtt tct tgt gaa act act tac ggt att act tta cgt gat act aag 48

Val Val Ser Cys Glu Thr Thr Tyr Gly Ile Thr Leu Arg Asp Thr Lys

1 5 10 15

gaa aaa ttc act gta ttc aaa gac ggt tcc gct gct act gat att gtt 96

Glu Lys Phe Thr Val Phe Lys Asp Gly Ser Ala Ala Thr Asp Ile Val

20 25 30

gaa tca gaa gat ggt tcc gtt tct tgg att gct act gct gcc ggt ggt 144

Glu Ser Glu Asp Gly Ser Val Ser Trp Ile Ala Thr Ala Ala Gly Gly

35 40 45

gct ggt ggt ggt gtt gcc ttc tat gtt aag gct aac aag gaa gaa att 192

Ala Gly Gly Gly Val Ala Phe Tyr Val Lys Ala Asn Lys Glu Glu Ile

50 55 60

aac att gct aac tat gaa tct atc gat att gaa atg gaa tac act cca 240

Asn Ile Ala Asn Tyr Glu Ser Ile Asp Ile Glu Met Glu Tyr Thr Pro

65 70 75 80

gtt gaa aac aaa tgg aat gat gct gct aag aac cca agt ttc tgt atg 288

Val Glu Asn Lys Trp Asn Asp Ala Ala Lys Asn Pro Ser Phe Cys Met

85 90 95

aga att ctt cca tgg gat tcc act ggt atg ttc ggt ggt tac gaa gat 336

Arg Ile Leu Pro Trp Asp Ser Thr Gly Met Phe Gly Gly Tyr Glu Asp

100 105 110

ctt gaa tac ttc gat act cca gca aaa tct ggt aat ttc aaa tac act 384

Leu Glu Tyr Phe Asp Thr Pro Ala Lys Ser Gly Asn Phe Lys Tyr Thr

115 120 125

att aag att cct tcc ttc ttt gct gat aag att tta tct agc tct gat 432

Ile Lys Ile Pro Ser Phe Phe Ala Asp Lys Ile Leu Ser Ser Ser Asp

130 135 140

ctc gat tct atc tta agt ttt gct atc aag ttc aac gat tat gaa aga 480

Leu Asp Ser Ile Leu Ser Phe Ala Ile Lys Phe Asn Asp Tyr Glu Arg

145 150 155 160

ggt aac acg gac ggt gac caa att aag att caa tta aag aat gtt aaa 528

Gly Asn Thr Asp Gly Asp Gln Ile Lys Ile Gln Leu Lys Asn Val Lys

165 170 175

ttc aac cca aag gaa aat gct cca gaa gat aag gct ttc gat gat ggt 576

Phe Asn Pro Lys Glu Asn Ala Pro Glu Asp Lys Ala Phe Asp Asp Gly

180 185 190

tta agg gat tct caa cgt ggt act gtc gtt gaa atg aaa tac tca tct 624

Leu Arg Asp Ser Gln Arg Gly Thr Val Val Glu Met Lys Tyr Ser Ser

195 200 205

aga gat tac acc gtc aag gaa tct gaa gct gac aaa tac gaa aag cac 672

Arg Asp Tyr Thr Val Lys Glu Ser Glu Ala Asp Lys Tyr Glu Lys His

210 215 220

gct tgg gtt tac ctt cca gct ggt tat gaa gct gat aac aag gat aag 720

Ala Trp Val Tyr Leu Pro Ala Gly Tyr Glu Ala Asp Asn Lys Asp Lys

225 230 235 240

aaa tac cca tta gtt gtt tta ctt cac ggt tat ggt caa aat gaa aac 768

Lys Tyr Pro Leu Val Val Leu Leu His Gly Tyr Gly Gln Asn Glu Asn

245 250 255

act tgg ggt ctt tcc aac aag ggt cgt ggt ggt aag atc aag ggt tac 816

Thr Trp Gly Leu Ser Asn Lys Gly Arg Gly Gly Lys Ile Lys Gly Tyr

260 265 270

atg gac aga ggt atg gct agt ggt aat gtt gaa aag ttt gtt ctt gtt 864

Met Asp Arg Gly Met Ala Ser Gly Asn Val Glu Lys Phe Val Leu Val

275 280 285

gcc gct act ggt gtt gcc agt aag aat tgg ggt cca aac ggt tct ggt 912

Ala Ala Thr Gly Val Ala Ser Lys Asn Trp Gly Pro Asn Gly Ser Gly

290 295 300

gtt gat ctt gat ggt ttc aat gct ttc ggt ggt gaa ctc aga aac gat 960

Val Asp Leu Asp Gly Phe Asn Ala Phe Gly Gly Glu Leu Arg Asn Asp

305 310 315 320

tta ctc cca tac att aga gct cac ttc aat gtt aag gtc gat cgt gat 1008

Leu Leu Pro Tyr Ile Arg Ala His Phe Asn Val Lys Val Asp Arg Asp

325 330 335

cac act gct tta gct ggt ctt tcc atg ggt ggt ggt caa act atc agt 1056

His Thr Ala Leu Ala Gly Leu Ser Met Gly Gly Gly Gln Thr Ile Ser

340 345 350

att ggt att ggt gaa act ctt gat gaa atc agt aac tac ggt tct ttc 1104

Ile Gly Ile Gly Glu Thr Leu Asp Glu Ile Ser Asn Tyr Gly Ser Phe

355 360 365

tct cca gct tta ttc caa act gct gaa gaa ttc ttc ggt aag gtt aag 1152

Ser Pro Ala Leu Phe Gln Thr Ala Glu Glu Phe Phe Gly Lys Val Lys

370 375 380

ggt aac ttc aag gaa gaa ctt aga att cac aac ctt tac atg act tgt 1200

Gly Asn Phe Lys Glu Glu Leu Arg Ile His Asn Leu Tyr Met Thr Cys

385 390 395 400

ggt gat gct gat act tta gtt tac gat act tac cca agt tac gtt gaa 1248

Gly Asp Ala Asp Thr Leu Val Tyr Asp Thr Tyr Pro Ser Tyr Val Glu

405 410 415

gct tta aag aat tgg gat gct gtt gaa ttc atg aag gaa tac act tac 1296

Ala Leu Lys Asn Trp Asp Ala Val Glu Phe Met Lys Glu Tyr Thr Tyr

420 425 430

cca ggt ggt act cac gat ttc cca gtt tgg tac aga ggt ttc aac gaa 1344

Pro Gly Gly Thr His Asp Phe Pro Val Trp Tyr Arg Gly Phe Asn Glu

435 440 445

ttc att caa att gtt ttc aaa aat caa aaa gtt aag gaa gaa cca att 1392

Phe Ile Gln Ile Val Phe Lys Asn Gln Lys Val Lys Glu Glu Pro Ile

450 455 460

cat gct gat cca gta gaa gac cca tct gat gaa cca gtt agt gtt gat 1440

His Ala Asp Pro Val Glu Asp Pro Ser Asp Glu Pro Val Ser Val Asp

465 470 475 480

cca tct gtt tct gtc gaa gaa cca aat gac agt gaa tct tcc tct gaa 1488

Pro Ser Val Ser Val Glu Glu Pro Asn Asp Ser Glu Ser Ser Ser Glu

485 490 495

gat gaa cca gtg gtt aaa aaa act att aag cac acc att gct aag aag 1536

Asp Glu Pro Val Val Lys Lys Thr Ile Lys His Thr Ile Ala Lys Lys

500 505 510

aag cca tct aag act aga act gtt acc aag aag gtc att aag aag aag 1584

Lys Pro Ser Lys Thr Arg Thr Val Thr Lys Lys Val Ile Lys Lys Lys

515 520 525

aat aac taagaaagtt tagttagtac agtagtgtaa aaaaaaaaaa aaaatcaaaa 1640

Asn Asn

530

agaaactcgt gccgaattcg at 1662

18

530

Orpinomyces sp. PC-2

18

Val Val Ser Cys Glu Thr Thr Tyr Gly Ile Thr Leu Arg Asp Thr Lys

1 5 10 15

Glu Lys Phe Thr Val Phe Lys Asp Gly Ser Ala Ala Thr Asp Ile Val

20 25 30

Glu Ser Glu Asp Gly Ser Val Ser Trp Ile Ala Thr Ala Ala Gly Gly

35 40 45

Ala Gly Gly Gly Val Ala Phe Tyr Val Lys Ala Asn Lys Glu Glu Ile

50 55 60

Asn Ile Ala Asn Tyr Glu Ser Ile Asp Ile Glu Met Glu Tyr Thr Pro

65 70 75 80

Val Glu Asn Lys Trp Asn Asp Ala Ala Lys Asn Pro Ser Phe Cys Met

85 90 95

Arg Ile Leu Pro Trp Asp Ser Thr Gly Met Phe Gly Gly Tyr Glu Asp

100 105 110

Leu Glu Tyr Phe Asp Thr Pro Ala Lys Ser Gly Asn Phe Lys Tyr Thr

115 120 125

Ile Lys Ile Pro Ser Phe Phe Ala Asp Lys Ile Leu Ser Ser Ser Asp

130 135 140

Leu Asp Ser Ile Leu Ser Phe Ala Ile Lys Phe Asn Asp Tyr Glu Arg

145 150 155 160

Gly Asn Thr Asp Gly Asp Gln Ile Lys Ile Gln Leu Lys Asn Val Lys

165 170 175

Phe Asn Pro Lys Glu Asn Ala Pro Glu Asp Lys Ala Phe Asp Asp Gly

180 185 190

Leu Arg Asp Ser Gln Arg Gly Thr Val Val Glu Met Lys Tyr Ser Ser

195 200 205

Arg Asp Tyr Thr Val Lys Glu Ser Glu Ala Asp Lys Tyr Glu Lys His

210 215 220

Ala Trp Val Tyr Leu Pro Ala Gly Tyr Glu Ala Asp Asn Lys Asp Lys

225 230 235 240

Lys Tyr Pro Leu Val Val Leu Leu His Gly Tyr Gly Gln Asn Glu Asn

245 250 255

Thr Trp Gly Leu Ser Asn Lys Gly Arg Gly Gly Lys Ile Lys Gly Tyr

260 265 270

Met Asp Arg Gly Met Ala Ser Gly Asn Val Glu Lys Phe Val Leu Val

275 280 285

Ala Ala Thr Gly Val Ala Ser Lys Asn Trp Gly Pro Asn Gly Ser Gly

290 295 300

Val Asp Leu Asp Gly Phe Asn Ala Phe Gly Gly Glu Leu Arg Asn Asp

305 310 315 320

Leu Leu Pro Tyr Ile Arg Ala His Phe Asn Val Lys Val Asp Arg Asp

325 330 335

His Thr Ala Leu Ala Gly Leu Ser Met Gly Gly Gly Gln Thr Ile Ser

340 345 350

Ile Gly Ile Gly Glu Thr Leu Asp Glu Ile Ser Asn Tyr Gly Ser Phe

355 360 365

Ser Pro Ala Leu Phe Gln Thr Ala Glu Glu Phe Phe Gly Lys Val Lys

370 375 380

Gly Asn Phe Lys Glu Glu Leu Arg Ile His Asn Leu Tyr Met Thr Cys

385 390 395 400

Gly Asp Ala Asp Thr Leu Val Tyr Asp Thr Tyr Pro Ser Tyr Val Glu

405 410 415

Ala Leu Lys Asn Trp Asp Ala Val Glu Phe Met Lys Glu Tyr Thr Tyr

420 425 430

Pro Gly Gly Thr His Asp Phe Pro Val Trp Tyr Arg Gly Phe Asn Glu

435 440 445

Phe Ile Gln Ile Val Phe Lys Asn Gln Lys Val Lys Glu Glu Pro Ile

450 455 460

His Ala Asp Pro Val Glu Asp Pro Ser Asp Glu Pro Val Ser Val Asp

465 470 475 480

Pro Ser Val Ser Val Glu Glu Pro Asn Asp Ser Glu Ser Ser Ser Glu

485 490 495

Asp Glu Pro Val Val Lys Lys Thr Ile Lys His Thr Ile Ala Lys Lys

500 505 510

Lys Pro Ser Lys Thr Arg Thr Val Thr Lys Lys Val Ile Lys Lys Lys

515 520 525

Asn Asn

530

19

400

Escherichia coli

19

Met Val Met Glu Leu Asn Glu Arg Asn Ile Thr Met Asn Ile Lys Ile

1 5 10 15

Ala Ala Leu Thr Leu Ala Ile Ala Ser Gly Ile Ser Ala Gln Trp Ala

20 25 30

Ile Ala Ala Asp Met Pro Ala Ser Pro Ala Pro Thr Ile Pro Val Lys

35 40 45

Gln Tyr Val Thr Gln Val Asn Ala Asp Asn Ser Val Thr Phe Arg Tyr

50 55 60

Phe Ala Pro Gly Ala Lys Asn Val Ser Val Val Val Gly Val Pro Val

65 70 75 80

Pro Asp Asn Ile His Pro Met Thr Lys Asp Glu Ala Gly Val Trp Ser

85 90 95

Trp Arg Thr Pro Ile Leu Lys Gly Asn Leu Tyr Glu Tyr Phe Phe Asn

100 105 110

Val Asp Gly Val Arg Ser Ile Asp Thr Gly Thr Ala Met Thr Asn Pro

115 120 125

Gln Arg Gln Val Asn Ser Ser Met Ile Leu Val Pro Gly Ser Tyr Leu

130 135 140

Asp Thr Arg Ser Val Ala His Gly Asp Leu Ile Ala Ile Thr Tyr His

145 150 155 160

Ser Asn Ala Leu Gln Ser Glu Arg Gln Met Tyr Val Trp Thr Pro Pro

165 170 175

Gly Tyr Thr Gly Met Gly Glu Pro Leu Pro Val Leu Tyr Phe Tyr His

180 185 190

Gly Phe Gly Asp Thr Gly Arg Ser Ala Ile Asp Gln Gly Arg Ile Pro

195 200 205

Gln Ile Met Asp Asn Leu Leu Ala Glu Gly Lys Ile Lys Pro Met Leu

210 215 220

Val Val Ile Pro Asp Thr Glu Thr Asp Ala Lys Gly Ile Ile Pro Glu

225 230 235 240

Asp Phe Val Pro Gln Glu Arg Arg Lys Val Phe Tyr Pro Leu Asn Ala

245 250 255

Lys Ala Ala Asp Arg Glu Leu Met Asn Asp Ile Ile Pro Leu Ile Ser

260 265 270

Lys Arg Phe Asn Val Arg Lys Asp Ala Asp Gly Arg Ala Leu Ala Gly

275 280 285

Leu Ser Gln Gly Gly Tyr Gln Ala Leu Val Ser Gly Met Asn His Leu

290 295 300

Glu Ser Phe Gly Trp Leu Ala Thr Phe Ser Gly Val Thr Thr Thr Thr

305 310 315 320

Val Pro Asp Glu Gly Val Ala Ala Arg Leu Asn Asp Pro Ala Ala Ile

325 330 335

Asn Gln Gln Leu Arg Asn Phe Thr Val Val Val Gly Asp Lys Asp Val

340 345 350

Val Thr Gly Lys Asp Ile Ala Gly Leu Lys Thr Glu Leu Glu Gln Lys

355 360 365

Lys Ile Asn Phe Asp Tyr Gln Glu Tyr Pro Gly Leu Asn His Glu Met

370 375 380

Asp Val Trp Arg Pro Ala Tyr Ala Ala Phe Val Gln Lys Leu Phe Lys

385 390 395 400

20

721

Aspergillus fumigatus

20

Met Gly Ala Phe Arg Trp Leu Ser Ile Ala Ala Ala Ala Ser Thr Ala

1 5 10 15

Leu Ala Leu Thr Pro Glu Gln Leu Ile Thr Ala Pro Arg Arg Ser Glu

20 25 30

Ala Ile Pro Asp Pro Ser Gly Lys Val Ala Val Phe Ser Thr Ser Gln

35 40 45

Tyr Ser Phe Glu Thr His Lys Arg Thr Ser Trp Trp Ser Leu Leu Asp

50 55 60

Leu Lys Thr Gly Gln Thr Lys Val Leu Thr Asn Asp Ser Ser Val Ser

65 70 75 80

Glu Ile Val Trp Leu Ser Asp Asp Ser Ile Leu Tyr Val Asn Ser Thr

85 90 95

Asn Ala Asp Ile Pro Gly Gly Val Glu Leu Trp Val Thr Gln Ala Ser

100 105 110

Ser Phe Ala Lys Gly Tyr Lys Ala Ala Ser Leu Pro Ala Ser Phe Ser

115 120 125

Gly Leu Lys Ala Ala Lys Thr Lys Ser Gly Asp Ile Arg Phe Val Ala

130 135 140

Tyr Gly Gln Ser Tyr Pro Asn Gly Thr Ala Tyr Asn Glu Glu Leu Ala

145 150 155 160

Thr Ala Pro Leu Ser Ser Ala Arg Ile Tyr Asp Ser Ile Tyr Val Arg

165 170 175

His Trp Asp Tyr Trp Leu Ser Thr Thr Phe Asn Ala Val Phe Ser Gly

180 185 190

Thr Leu Lys Lys Gly His Gly Lys Asn Gly Tyr Ser Leu Asp Gly Glu

195 200 205

Leu Lys Asn Leu Val Ser Pro Val Lys Asn Ala Glu Ser Pro Tyr Pro

210 215 220

Pro Phe Gly Gly Ala Ser Asp Tyr Asp Leu Ser Pro Asp Gly Lys Trp

225 230 235 240

Val Ala Phe Lys Ser Lys Ala Pro Glu Leu Pro Lys Ala Asn Phe Thr

245 250 255

Thr Ser Tyr Ile Tyr Leu Val Pro His Asp Ala Ser Glu Thr Ala Arg

260 265 270

Pro Ile Asn Gly Pro Asp Ser Pro Gly Thr Pro Lys Gly Ile Lys Gly

275 280 285

Asp Ser Ser Ser Pro Val Phe Ser Pro Asn Gly Asp Lys Leu Ala Tyr

290 295 300

Phe Gln Met Arg Asp Glu Thr Tyr Glu Ser Asp Arg Ala Leu Leu Tyr

305 310 315 320

Val Tyr Ser Leu Gly Ser Lys Lys Thr Ile Pro Ser Val Ala Gly Asp

325 330 335

Trp Asp Arg Ser Pro Asp Ser Val Lys Trp Thr Pro Asp Gly Lys Thr

340 345 350

Leu Ile Val Gly Ser Glu Asp Leu Gly Arg Thr Arg Leu Phe Ser Leu

355 360 365

Pro Ala Asn Ala Lys Asp Asp Tyr Lys Pro Lys Asn Phe Thr Asp Gly

370 375 380

Gly Ser Val Ser Ala Tyr Tyr Phe Leu Pro Asp Ser Ser Leu Leu Val

385 390 395 400

Thr Gly Ser Ala Leu Trp Thr Asn Trp Asn Val Tyr Thr Ala Lys Pro

405 410 415

Glu Lys Gly Val Ile Lys Lys Ile Ala Ser Ala Asn Glu Ile Asp Pro

420 425 430

Glu Leu Lys Gly Leu Gly Pro Ser Asp Ile Ser Glu Phe Tyr Phe Gln

435 440 445

Gly Asn Phe Thr Asp Ile His Ala Trp Val Ile Tyr Pro Glu Asn Phe

450 455 460

Asp Lys Ser Lys Lys Tyr Pro Leu Ile Phe Phe Ile His Gly Gly Pro

465 470 475 480

Gln Gly Asn Trp Ala Asp Gly Trp Ser Thr Arg Trp Asn Pro Lys Ala

485 490 495

Trp Ala Asp Gln Gly Tyr Val Val Val Ala Pro Asn Pro Thr Gly Ser

500 505 510

Thr Gly Phe Gly Gln Ala Leu Thr Thr Ala Ile Gln Asn Asn Trp Gly

515 520 525

Gly Ala Pro Tyr Asp Asp Leu Val Lys Cys Trp Glu Tyr Val His Glu

530 535 540

Asn Leu Asp Tyr Val Asp Thr Asp His Gly Val Ala Ala Gly Ala Ser

545 550 555 560

Tyr Gly Gly Phe Met Ile Asn Trp Ile Gln Gly Ser Pro Leu Gly Arg

565 570 575

Lys Phe Lys Ala Leu Val Ser His Asp Gly Thr Phe Val Ala Asp Ala

580 585 590

Lys Val Ser Thr Glu Glu Leu Trp Phe Met Gln Arg Glu Phe Asn Gly

595 600 605

Thr Phe Trp Asp Ala Arg Asp Asn Tyr Arg Arg Trp Asp Pro Ser Ala

610 615 620

Pro Glu Arg Ile Leu Gln Phe Ala Thr Pro Met Leu Val Ile His Ser

625 630 635 640

Asp Lys Asp Tyr Arg Leu Pro Val Ala Glu Gly Leu Ser Leu Phe Asn

645 650 655

Val Leu Gln Glu Arg Gly Val Pro Ser Arg Phe Leu Asn Phe Pro Asp

660 665 670

Glu Asn His Trp Val Val Asn Pro Glu Asn Ser Leu Val Trp His Gln

675 680 685

Gln Ala Leu Gly Trp Ile Asn Lys Tyr Ser Gly Val Glu Lys Ser Asn

690 695 700

Pro Asn Ala Val Ser Leu Glu Asp Thr Val Val Pro Val Val Asn Tyr

705 710 715 720

Asn

21

20

Orpinomyces sp. PC-2

Description of Artificial SequenceN-terminal

amino acid sequence of a feruloyl esterase of

Orpinomyces PC-2.

21

Glu Thr Thr Tyr Gly Ile Thr Leu Arg Asp Thr Lys Glu Lys Phe Thr

1 5 10 15

Val Phe Lys Asp

20

22

400

Escherichia coli

22

Met Val Met Glu Leu Asn Glu Arg Asn Ile Thr Met Asn Ile Lys Ile

1 5 10 15

Ala Ala Leu Thr Leu Ala Ile Ala Ser Gly Ile Ser Ala Gln Trp Ala

20 25 30

Ile Ala Ala Asp Met Pro Ala Ser Pro Ala Pro Thr Ile Pro Val Lys

35 40 45

Gln Tyr Val Thr Gln Val Asn Ala Asp Asn Ser Val Thr Phe Arg Tyr

50 55 60

Phe Ala Pro Gly Ala Lys Asn Val Ser Val Val Val Gly Val Pro Val

65 70 75 80

Pro Asp Asn Ile His Pro Met Thr Lys Asp Glu Ala Gly Val Trp Ser

85 90 95

Trp Arg Thr Pro Ile Leu Lys Gly Asn Leu Tyr Glu Tyr Phe Phe Asn

100 105 110

Val Asp Gly Val Arg Ser Ile Asp Thr Gly Thr Ala Met Thr Asn Pro

115 120 125

Gln Arg Gln Val Asn Ser Ser Met Ile Leu Val Pro Gly Ser Tyr Leu

130 135 140

Asp Thr Arg Ser Val Ala His Gly Asp Leu Ile Ala Ile Thr Tyr His

145 150 155 160

Ser Asn Ala Leu Gln Ser Glu Arg Gln Met Tyr Val Trp Thr Pro Pro

165 170 175

Gly Tyr Thr Gly Met Gly Glu Pro Leu Pro Val Leu Tyr Phe Tyr His

180 185 190

Gly Phe Gly Asp Thr Gly Arg Ser Ala Ile Asp Gln Gly Arg Ile Pro

195 200 205

Gln Ile Met Asp Asn Leu Leu Ala Glu Gly Lys Ile Lys Pro Met Leu

210 215 220

Val Val Ile Pro Asp Thr Glu Thr Asp Ala Lys Gly Ile Ile Pro Glu

225 230 235 240

Asp Phe Val Pro Gln Glu Arg Arg Lys Val Phe Tyr Pro Leu Asn Ala

245 250 255

Lys Ala Ala Asp Arg Glu Leu Met Asn Asp Ile Ile Pro Leu Ile Ser

260 265 270

Lys Arg Phe Asn Val Arg Lys Asp Ala Asp Gly Arg Ala Leu Ala Gly

275 280 285

Leu Ser Gln Gly Gly Tyr Gln Ala Leu Val Ser Gly Met Asn His Leu

290 295 300

Glu Ser Phe Gly Trp Leu Ala Thr Phe Ser Gly Val Thr Thr Thr Thr

305 310 315 320

Val Pro Asp Glu Gly Val Ala Ala Arg Leu Asn Asp Pro Ala Ala Ile

325 330 335

Asn Gln Gln Leu Arg Asn Phe Thr Val Val Val Gly Asp Lys Asp Val

340 345 350

Val Thr Gly Lys Asp Ile Ala Gly Leu Lys Thr Glu Leu Glu Gln Lys

355 360 365

Lys Ile Asn Phe Asp Tyr Gln Glu Tyr Pro Gly Leu Asn His Glu Met

370 375 380

Asp Val Trp Arg Pro Ala Tyr Ala Ala Phe Val Gln Lys Leu Phe Lys

385 390 395 400

23

aagcttaatt tgtttggtat accttgcttt atgttcaatc acgttctcgt cattaaacaa 60

cccatataag ctgctccctg accggaaagt tgaacattga ttcttgcatt ccgaatctgc 120

tccaataaaa catttctgaa tttcgagacg gcaaaaaatg atgccgcttc catttcaaca 180

gtaacacagc cttctgcaat ccttttcgtc agcttccttt aaattttaag tttgtctatt 240

gacaaaacta aaaactgtaa ttactataaa aatataacta ataaattaca tttttaacat 300

cattatgggg tactggtaaa gacgtgatag ttattaataa atttaacaaa taataacaca 360

ctgctatctt cgaccgtaaa tttactatgt ctctaatgta atatgacata aataatataa 420

gtaaaggagg taaaagttt atg aag cgt aag gtt aag aag atg gca gct atg 472

Met Lys Arg Lys Val Lys Lys Met Ala Ala Met

1 5 10

gca acg agt ata att atg gct atc atg atc atc cta cat agt ata cca 520

Ala Thr Ser Ile Ile Met Ala Ile Met Ile Ile Leu His Ser Ile Pro

15 20 25

gta ctc gcc ggg cga ata att tac gac aat gag aca ggc aca cat gga 568

Val Leu Ala Gly Arg Ile Ile Tyr Asp Asn Glu Thr Gly Thr His Gly

30 35 40

ggc tac gac tat gag ctc tgg aaa gac tac gga aat acg att atg gaa 616

Gly Tyr Asp Tyr Glu Leu Trp Lys Asp Tyr Gly Asn Thr Ile Met Glu

45 50 55

ctt aac gac ggt ggt act ttt agt tgt caa tgg agt aat atc ggt aat 664

Leu Asn Asp Gly Gly Thr Phe Ser Cys Gln Trp Ser Asn Ile Gly Asn

60 65 70 75

gca cta ttt aga aaa ggg aga aaa ttt aat tcc gac aaa acc tat caa 712

Ala Leu Phe Arg Lys Gly Arg Lys Phe Asn Ser Asp Lys Thr Tyr Gln

80 85 90

gaa tta gga gac ata gta gtt gaa tat ggc tgt gat tac aat cca aac 760

Glu Leu Gly Asp Ile Val Val Glu Tyr Gly Cys Asp Tyr Asn Pro Asn

95 100 105

gga aat tcc tat ttg tgt gtt tac ggt tgg aca aga aat cca ctg gtt 808

Gly Asn Ser Tyr Leu Cys Val Tyr Gly Trp Thr Arg Asn Pro Leu Val

110 115 120

gaa tat tac att gta gaa agc tgg ggc agc tgg cgt cca cct gga gca 856

Glu Tyr Tyr Ile Val Glu Ser Trp Gly Ser Trp Arg Pro Pro Gly Ala

125 130 135

aca ccc aaa gga acc atc aca cag tgg atg gca ggt act tat gaa ata 904

Thr Pro Lys Gly Thr Ile Thr Gln Trp Met Ala Gly Thr Tyr Glu Ile

140 145 150 155

tat gaa act acc cgg gta aat cag cct tcc atc gat gga act gcg aca 952

Tyr Glu Thr Thr Arg Val Asn Gln Pro Ser Ile Asp Gly Thr Ala Thr

160 165 170

ttc caa caa tat tgg agt gtt cgt aca tcc aag aga aca agc gga aca 1000

Phe Gln Gln Tyr Trp Ser Val Arg Thr Ser Lys Arg Thr Ser Gly Thr

175 180 185

ata tct gtc act gaa cat ttt aaa cag tgg gaa aga atg ggc atg cga 1048

Ile Ser Val Thr Glu His Phe Lys Gln Trp Glu Arg Met Gly Met Arg

190 195 200

atg ggt aag atg tat gaa gtt gct ctt acc gtt gaa ggt tat cag agc 1096

Met Gly Lys Met Tyr Glu Val Ala Leu Thr Val Glu Gly Tyr Gln Ser

205 210 215

agt ggg tac gct aat gta tac aag aat gaa atc aga ata ggt gca aat 1144

Ser Gly Tyr Ala Asn Val Tyr Lys Asn Glu Ile Arg Ile Gly Ala Asn

220 225 230 235

cca act cct gcc cca tct caa agc cca att aga aga gat gca ttt tca 1192

Pro Thr Pro Ala Pro Ser Gln Ser Pro Ile Arg Arg Asp Ala Phe Ser

240 245 250

ata atc gaa gcg gaa gaa tat aac agc aca aat tcc tcc act tta caa 1240

Ile Ile Glu Ala Glu Glu Tyr Asn Ser Thr Asn Ser Ser Thr Leu Gln

255 260 265

gtg att gga acg cca aat aat ggc aga gga att ggt tat att gaa aat 1288

Val Ile Gly Thr Pro Asn Asn Gly Arg Gly Ile Gly Tyr Ile Glu Asn

270 275 280

ggt aat acc gta act tac agc aat ata gat ttt ggt agt ggt gca aca 1336

Gly Asn Thr Val Thr Tyr Ser Asn Ile Asp Phe Gly Ser Gly Ala Thr

285 290 295

ggg ttc tct gca act gtt gca acg gag gtt aat acc tca att caa atc 1384

Gly Phe Ser Ala Thr Val Ala Thr Glu Val Asn Thr Ser Ile Gln Ile

300 305 310 315

cgt tct gac agt cct acc gga act cta ctt ggt acc tta tat gta agt 1432

Arg Ser Asp Ser Pro Thr Gly Thr Leu Leu Gly Thr Leu Tyr Val Ser

320 325 330

tct acc ggc agc tgg aat aca tat caa acc gta tct aca aac atc agc 1480

Ser Thr Gly Ser Trp Asn Thr Tyr Gln Thr Val Ser Thr Asn Ile Ser

335 340 345

aaa att acc ggc gtt cat gat att gta ttg gta ttc tca ggt cca gtc 1528

Lys Ile Thr Gly Val His Asp Ile Val Leu Val Phe Ser Gly Pro Val

350 355 360

aat gtg gac aac ttc ata ttt agc aga agt tca cca gtg cct gca cct 1576

Asn Val Asp Asn Phe Ile Phe Ser Arg Ser Ser Pro Val Pro Ala Pro

365 370 375

ggt gat aac aca aga gac gca tat tct atc att cag gcc gag gat tat 1624

Gly Asp Asn Thr Arg Asp Ala Tyr Ser Ile Ile Gln Ala Glu Asp Tyr

380 385 390 395

gac agc agt tat ggt ccc aac ctt caa atc ttt agc tta cca ggt ggt 1672

Asp Ser Ser Tyr Gly Pro Asn Leu Gln Ile Phe Ser Leu Pro Gly Gly

400 405 410

ggc agc gcc att ggc tat att gaa aat ggt tat tcc act acc tat aaa 1720

Gly Ser Ala Ile Gly Tyr Ile Glu Asn Gly Tyr Ser Thr Thr Tyr Lys

415 420 425

aat att gat ttt ggt gac ggc gca acg tcc gta aca gca aga gta gct 1768

Asn Ile Asp Phe Gly Asp Gly Ala Thr Ser Val Thr Ala Arg Val Ala

430 435 440

acc cag aat gct act acc att cag gta aga ttg gga agt cca tcg ggt 1816

Thr Gln Asn Ala Thr Thr Ile Gln Val Arg Leu Gly Ser Pro Ser Gly

445 450 455

aca tta ctt gga aca att tac gtg ggg tcc aca gga agc ttt gat act 1864

Thr Leu Leu Gly Thr Ile Tyr Val Gly Ser Thr Gly Ser Phe Asp Thr

460 465 470 475

tat agg gat gta tcc gct acc att agt aat act gcg ggt gta aaa gat 1912

Tyr Arg Asp Val Ser Ala Thr Ile Ser Asn Thr Ala Gly Val Lys Asp

480 485 490

att gtt ctt gta ttc tca ggt cct gtt aat gtt gac tgg ttt gta ttc 1960

Ile Val Leu Val Phe Ser Gly Pro Val Asn Val Asp Trp Phe Val Phe

495 500 505

tca aaa tca gga act taagggtata gaccctaatg tggagtacaa aatctggtat 2015

Ser Lys Ser Gly Thr

510

ggcatatata aaaaaagact tggaattgta ccagtgcgac atataatggc tttgtaaaat 2075

attctgatta aaacggaatg tttaaggata ggaaaagaaa gtattctttt cctgtctttt 2135

ttatgtaacc ttaaaaatta cagccaatta ttcaataaaa taatttctgt aaatcagtta 2195

ttcttgaacc aatattaaaa gaatttcccc aaggtcttta atgtctggcc ggattacatt 2255

atcttctcct gtcattttaa aaaacagtta aatcaagctt ttgtcgcaat agaatgaatt 2315

attatttggg attccaaacc aaagacatat cattaagcag ttgtaaaaa 2364

24

512

Clostridium stercorarium

24

Met Lys Arg Lys Val Lys Lys Met Ala Ala Met Ala Thr Ser Ile Ile

1 5 10 15

Met Ala Ile Met Ile Ile Leu His Ser Ile Pro Val Leu Ala Gly Arg

20 25 30

Ile Ile Tyr Asp Asn Glu Thr Gly Thr His Gly Gly Tyr Asp Tyr Glu

35 40 45

Leu Trp Lys Asp Tyr Gly Asn Thr Ile Met Glu Leu Asn Asp Gly Gly

50 55 60

Thr Phe Ser Cys Gln Trp Ser Asn Ile Gly Asn Ala Leu Phe Arg Lys

65 70 75 80

Gly Arg Lys Phe Asn Ser Asp Lys Thr Tyr Gln Glu Leu Gly Asp Ile

85 90 95

Val Val Glu Tyr Gly Cys Asp Tyr Asn Pro Asn Gly Asn Ser Tyr Leu

100 105 110

Cys Val Tyr Gly Trp Thr Arg Asn Pro Leu Val Glu Tyr Tyr Ile Val

115 120 125

Glu Ser Trp Gly Ser Trp Arg Pro Pro Gly Ala Thr Pro Lys Gly Thr

130 135 140

Ile Thr Gln Trp Met Ala Gly Thr Tyr Glu Ile Tyr Glu Thr Thr Arg

145 150 155 160

Val Asn Gln Pro Ser Ile Asp Gly Thr Ala Thr Phe Gln Gln Tyr Trp

165 170 175

Ser Val Arg Thr Ser Lys Arg Thr Ser Gly Thr Ile Ser Val Thr Glu

180 185 190

His Phe Lys Gln Trp Glu Arg Met Gly Met Arg Met Gly Lys Met Tyr

195 200 205

Glu Val Ala Leu Thr Val Glu Gly Tyr Gln Ser Ser Gly Tyr Ala Asn

210 215 220

Val Tyr Lys Asn Glu Ile Arg Ile Gly Ala Asn Pro Thr Pro Ala Pro

225 230 235 240

Ser Gln Ser Pro Ile Arg Arg Asp Ala Phe Ser Ile Ile Glu Ala Glu

245 250 255

Glu Tyr Asn Ser Thr Asn Ser Ser Thr Leu Gln Val Ile Gly Thr Pro

260 265 270

Asn Asn Gly Arg Gly Ile Gly Tyr Ile Glu Asn Gly Asn Thr Val Thr

275 280 285

Tyr Ser Asn Ile Asp Phe Gly Ser Gly Ala Thr Gly Phe Ser Ala Thr

290 295 300

Val Ala Thr Glu Val Asn Thr Ser Ile Gln Ile Arg Ser Asp Ser Pro

305 310 315 320

Thr Gly Thr Leu Leu Gly Thr Leu Tyr Val Ser Ser Thr Gly Ser Trp

325 330 335

Asn Thr Tyr Gln Thr Val Ser Thr Asn Ile Ser Lys Ile Thr Gly Val

340 345 350

His Asp Ile Val Leu Val Phe Ser Gly Pro Val Asn Val Asp Asn Phe

355 360 365

Ile Phe Ser Arg Ser Ser Pro Val Pro Ala Pro Gly Asp Asn Thr Arg

370 375 380

Asp Ala Tyr Ser Ile Ile Gln Ala Glu Asp Tyr Asp Ser Ser Tyr Gly

385 390 395 400

Pro Asn Leu Gln Ile Phe Ser Leu Pro Gly Gly Gly Ser Ala Ile Gly

405 410 415

Tyr Ile Glu Asn Gly Tyr Ser Thr Thr Tyr Lys Asn Ile Asp Phe Gly

420 425 430

Asp Gly Ala Thr Ser Val Thr Ala Arg Val Ala Thr Gln Asn Ala Thr

435 440 445

Thr Ile Gln Val Arg Leu Gly Ser Pro Ser Gly Thr Leu Leu Gly Thr

450 455 460

Ile Tyr Val Gly Ser Thr Gly Ser Phe Asp Thr Tyr Arg Asp Val Ser

465 470 475 480

Ala Thr Ile Ser Asn Thr Ala Gly Val Lys Asp Ile Val Leu Val Phe

485 490 495

Ser Gly Pro Val Asn Val Asp Trp Phe Val Phe Ser Lys Ser Gly Thr

500 505 510

25

ggcacgagga aatttttttt actggttaaa aaaaaattat aaaactaaat aaataaaaaa 60

aatatttttt gaaatatatt aaaataggaa aaaaaa atg aga act att aaa ttt 114

Met Arg Thr Ile Lys Phe

1 5

tta ttc gca tta gct att aca acc gtt gct aag gcc caa tgg ggt gga 162

Leu Phe Ala Leu Ala Ile Thr Thr Val Ala Lys Ala Gln Trp Gly Gly

10 15 20

aac ggt ggt gcc tct gct ggt caa aga tta agc gtt ggt ggt ggt caa 210

Asn Gly Gly Ala Ser Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln

25 30 35

aac caa cat aaa ggt gtt ttt gat ggc ttc agt tat gaa atc tgg tta 258

Asn Gln His Lys Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Leu

40 45 50

gat aac acc ggt ggt agt ggt tcc atg acc ctt ggt aaa ggt gca acc 306

Asp Asn Thr Gly Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr

55 60 65 70

ttc aag gct gaa tgg agt gca gct gtt aac cgt ggt aac ttc ctt gcc 354

Phe Lys Ala Glu Trp Ser Ala Ala Val Asn Arg Gly Asn Phe Leu Ala

75 80 85

cgt cgt ggt ctt gat ttc ggt tct acc aaa aag gca acc gct tac gaa 402

Arg Arg Gly Leu Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu

90 95 100

tac atc gga ttg gat tat gaa gca agt tac aga caa act gcc agc gca 450

Tyr Ile Gly Leu Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala

105 110 115

agt ggt aac tcc cgt ctt tgt gta tac ggc tgg ttc caa aac cgt gga 498

Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly

120 125 130

gtt caa ggc gta cct ttg gta gaa tac tac atc att gaa gat tgg gtt 546

Val Gln Gly Val Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val

135 140 145 150

gac tgg gta cca gat gca caa gga aaa atg gta acc atc gat ggt gca 594

Asp Trp Val Pro Asp Ala Gln Gly Lys Met Val Thr Ile Asp Gly Ala

155 160 165

caa tat aag att ttc caa atg gat cac act ggt cca act atc aat ggt 642

Gln Tyr Lys Ile Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly

170 175 180

ggt aat gaa acc ttt aag caa tac ttc agt gtc cgt caa caa aag aga 690

Gly Asn Glu Thr Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg

185 190 195

act tct ggt cat att act gta tca gat cac ttt aag gca tgg tcc aat 738

Thr Ser Gly His Ile Thr Val Ser Asp His Phe Lys Ala Trp Ser Asn

200 205 210

caa ggt tgg ggt att gga aac ctc tat gaa gtt gca ttg aac gca gaa 786

Gln Gly Trp Gly Ile Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu

215 220 225 230

ggt tgg caa agt agt ggt gtc gct gac gtc ccc aag ttg gat gtc tac 834

Gly Trp Gln Ser Ser Gly Val Ala Asp Val Pro Lys Leu Asp Val Tyr

235 240 245

acc acc aaa caa ggt tct gct cct cgt act acc acc acc act acc cgt 882

Thr Thr Lys Gln Gly Ser Ala Pro Arg Thr Thr Thr Thr Thr Thr Arg

250 255 260

act act acc cgt act act aca aaa aca ctt cca acc act aat aaa aaa 930

Thr Thr Thr Arg Thr Thr Thr Lys Thr Leu Pro Thr Thr Asn Lys Lys

265 270 275

tgt tct gcc aag att act gcc caa ggt tac aag tgt tgt agt gat cca 978

Cys Ser Ala Lys Ile Thr Ala Gln Gly Tyr Lys Cys Cys Ser Asp Pro

280 285 290

aat tgt gtt gtt tac tac act gat gaa gat ggt acc tgg ggt gtt gaa 1026

Asn Cys Val Val Tyr Tyr Thr Asp Glu Asp Gly Thr Trp Gly Val Glu

295 300 305 310

aac aat caa tgg tgt gga tgt ggt gtt gaa gca tgt tct ggc aag att 1074

Asn Asn Gln Trp Cys Gly Cys Gly Val Glu Ala Cys Ser Gly Lys Ile

315 320 325

act gcc caa ggt tac aag tgt tgt agt gat cca aag tgt gtt gtt tac 1122

Thr Ala Gln Gly Tyr Lys Cys Cys Ser Asp Pro Lys Cys Val Val Tyr

330 335 340

tac act gat gac gat ggt aaa tgg ggt gtt gaa aac aac gaa tgg tgt 1170

Tyr Thr Asp Asp Asp Gly Lys Trp Gly Val Glu Asn Asn Glu Trp Cys

345 350 355

ggt tgt ggt tta taagcagaaa aatactaatt tagtaaaaaa aaaaaaaaa 1221

Gly Cys Gly Leu

360

26

362

Orpinomyces sp. PC-2

26

Met Arg Thr Ile Lys Phe Leu Phe Ala Leu Ala Ile Thr Thr Val Ala

1 5 10 15

Lys Ala Gln Trp Gly Gly Asn Gly Gly Ala Ser Ala Gly Gln Arg Leu

20 25 30

Ser Val Gly Gly Gly Gln Asn Gln His Lys Gly Val Phe Asp Gly Phe

35 40 45

Ser Tyr Glu Ile Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser Met Thr

50 55 60

Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu Trp Ser Ala Ala Val Asn

65 70 75 80

Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Thr Lys

85 90 95

Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu Asp Tyr Glu Ala Ser Tyr

100 105 110

Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly

115 120 125

Trp Phe Gln Asn Arg Gly Val Gln Gly Val Pro Leu Val Glu Tyr Tyr

130 135 140

Ile Ile Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gln Gly Lys Met

145 150 155 160

Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile Phe Gln Met Asp His Thr

165 170 175

Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr Phe Lys Gln Tyr Phe Ser

180 185 190

Val Arg Gln Gln Lys Arg Thr Ser Gly His Ile Thr Val Ser Asp His

195 200 205

Phe Lys Ala Trp Ser Asn Gln Gly Trp Gly Ile Gly Asn Leu Tyr Glu

210 215 220

Val Ala Leu Asn Ala Glu Gly Trp Gln Ser Ser Gly Val Ala Asp Val

225 230 235 240

Pro Lys Leu Asp Val Tyr Thr Thr Lys Gln Gly Ser Ala Pro Arg Thr

245 250 255

Thr Thr Thr Thr Thr Arg Thr Thr Thr Arg Thr Thr Thr Lys Thr Leu

260 265 270

Pro Thr Thr Asn Lys Lys Cys Ser Ala Lys Ile Thr Ala Gln Gly Tyr

275 280 285

Lys Cys Cys Ser Asp Pro Asn Cys Val Val Tyr Tyr Thr Asp Glu Asp

290 295 300

Gly Thr Trp Gly Val Glu Asn Asn Gln Trp Cys Gly Cys Gly Val Glu

305 310 315 320

Ala Cys Ser Gly Lys Ile Thr Ala Gln Gly Tyr Lys Cys Cys Ser Asp

325 330 335

Pro Lys Cys Val Val Tyr Tyr Thr Asp Asp Asp Gly Lys Trp Gly Val

340 345 350

Glu Asn Asn Glu Trp Cys Gly Cys Gly Leu

355 360